Functionalization of Spider Silk with Affinity and Bioactive Domains Via Genetic Engineering for in Vitro Disease Diagnosis and Tissue Engineering

Functionalization of spider silk with affinity and bioactive domains via genetic engineering for in vitro disease diagnosis and tissue engineering

Naresh Thatikonda

Kungliga Tekniska Högskolan, KTH Royal Institute of Technology School of Engineering Sciences in Chemistry, Biotechnology and Health Stockholm, Sweden 2018

KTH Royal Institute of Technology School of Engineering Science in Chemistry, Biotechnology and Health Department of Protein Science AlbaNova University Center SE-106 91 Stockholm Sweden

Cover image: Representation of functionalized spider silk for its use in disease diagnosis and tissue engineering.

Printed by Universitetsservice, US-AB, 2018

ISBN 978-91-7729-970-7 TRITA-CBH-FOU-2018:48

Abstract In the recent past, spider silk has drawn significant attention from researchers mainly due to its distinguished mechanical strength, elasticity, biocompatibility and biodegradability. Technological advancements in genetic engineering have resulted in methods for creation of partial spider silk proteins. The main objective of this thesis has been to functionalize a partial spider silk protein, 4RepCT, with different affinity and bioactive domains via genetic engineering. Furthermore, the applicability of materials based on functionalized/bioactivated partial spider silk proteins for in vitro disease diagnosis and tissue engineering applications has been investigated. In Paper I, four affinity domains of different sizes and folds were genetically attached to 4RepCT. All four silk fusion proteins could self-assemble to silk-like fibers. The retained ability of each added affinity domain to bind its respective target while in silk format was also verified. A construct where a monomeric streptavidin domain was genetically fused to 4RepCT was used to allow non-covalent presentation of biotinylated growth factors. Such materials have potential for applications where capture of growth factors could be advantageous, for example in vitro cell culture studies. In Paper II, as a proof-of-concept, two recombinant antibody fragments (scFvs), previously shown to contribute to the candidate protein signature for diagnosing Systemic Lupus Erythematosus (SLE), were covalently attached to either ends of two types of partial spider silk proteins, 4RepCT and NTCT. All of the generated silk fusion proteins were shown able to self-assemble into fibres as well as defined spots in an array. Significantly higher target detection signal was reported from scFv-silk fusion proteins when compared to the same added amount of scFvs alone in micro- and nanoarrays. Thus, scFv-silk fusion proteins can be used as capture probes in the generation of sensitive diagnostic immunoassays for effective disease diagnosis. In Paper III, bioactivation of 4RepCT with a pleiotropic growth factor, basic fibroblast growth factor (bFGF) was investigated. The generated silk-bFGF fusion protein retained the propensity to self-assemble into surface coatings and silk-like fibers. Maintained functionality of the silk-bFGF coating to bind FGFR receptor was confirmed using surface plasmon resonance studies. Moreover, with the aim to create an artificial ECM, silk-bFGF protein was combined with FN-silk, an engineered spider silk protein previously reported to support cell adhesion. Retained bioactivity of the bFGF was confirmed by culture of primary human endothelial cells on combined silk coatings and within combined silk fibers, even when cultured in medium containing low serum and no supplemented soluble growth factors. These findings highlight the use of combined silk coatings for in vitro cell culture, and combined silk fibers as a potential scaffold for tissue engineering applications. In Paper IV, the possibility to genetically fuse two affibody-based VEGFR2 binders,

Zdimer and Ztetramer, to 4RepCT was investigated. Maintained activity of added engineered

i affibodies was confirmed by receptor phosphorylation and cell proliferation studies. Furthermore, the possibility to create vessel-like structures within a FN-silk based cell scaffold containing Ztetramer-silk fibrils was reported. These findings highlight the future potential of herein developed silk based cell scaffolds in regenerative medicine.

Keywords: affinity domains, artificial ECM, basic fibroblast growth factor, functionalization, mammalian cell culture, micro-and nano arrays, partial spider silk, single chain variable fragments.

Author’s address: Naresh Thatikonda, Department of Protein Science, School of Engineering Sciences in Chemistry, Biotechnology and Health, KTH Royal Institute of Technology, Stockholm, Sweden E-mail: [email protected]

ii Abstract in Swedish Under senare tid har silke ifrån spindlar fått stor uppmärksamhet från forskare, främst på grund av sin mekaniska styrka, elasticitet, biokompatibilitet och biologisk nedbrytbarhet. Tekniska framsteg inom genteknik har resulterat i metoder för att designa konstgjorda spindelsilkesproteiner. Huvudsyftet med denna avhandling har varit att funktionalisera det designade spindelsilkesproteinet, 4RepCT, med olika affinitets- och bioaktiva domäner med hjälp av genteknik. Vidare har användbarheten av material baserade på dessa funktionaliserade/bioaktiverade spindelsilkesproteiner för in vitro-sjukdomsdiagnostik och vävnadstekniska tillämpningar undersökts. I artikel I, har fyra affinitetsdomäner av olika storlekar och veckning genetiskt kopplas ihop med 4RepCT. Alla fyra silkesfusionsproteiner kunde bilda silkesliknande fibrer. Den behållna förmågan hos varje tillförd affinitetsdomän för att binda dess respektive målmolekyl i silkesformatet verifierades också. En konstruktion där en monomer streptavidindomän var genetiskt kopplad till 4RepCT användes för att tillåta icke-kovalent presentation av biotinylerade tillväxtfaktorer. Sådana material har potential för tillämpningar där infångning av tillväxtfaktorer kan vara fördelaktig, exempelvis i in vitro-cellodlingsstudier. I aritkel II, användes två rekombinanta antikroppsfragment (scFvs), som tidigare visat sig bidra till proteinsignaturen för diagnostisering av systemisk lupus erytematosus (SLE), kovalent bundna till ändarna av två typer av designade spindelsilkesproteiner, 4RepCT och NTCT. Alla de genererade silkesfusionsproteinerna visade sig kunna bilda i fibrer såväl som definierade proteinspottar i en array. Signifikant högre måldetekteringssignal rapporterades från scFv-silkesfusionsproteinerna jämfört med samma tillsatta mängd av ensamma scFvs i mikro- och nanoarrayer. ScFv-silkesfusionsproteiner skulle därfor kunna användas som infångningsprober för generering av känsliga immunoanalyser för effektiv sjukdomsdiagnos. I artikel III, undersöktes bioaktivering av 4RepCT med en pleiotrop tillväxtfaktor, fibroblasttillväxtfaktorn, bFGF. Det genererade silke-bFGF-fusionsproteinet behöll benägenheten att bilda ytbeläggningar och silkesliknande fibrer. Bibehållen funktionalitet hos silk-bFGF för att binda FGFR-receptor bekräftades med användning av ytplasmonresonansstudier. I syfte att skapa en artificiell extracellular matrix, kombinerades silke-bFGF-proteinet med FN-silke, ett designat spindelsilkesprotein som tidigare visat sig främja celladhesion. Bibehållen bioaktivitet hos bFGF bekräftades genom odling av primära humana endotelceller på bade silkecoating och silkefibrer av silk-bFGF, även när de odlades i medium innehållande låg serumhalt och utan tillsats av lösliga tillväxtfaktorer. Dessa resultat visar på potentialen för användning inom in vitro-cellodling och resultat visar på potentialen för silkesfibrer som en byggnadsställning för tillämpningar inom tissue engineering. I artikel IV, undersöktes möjligheten att genetiskt sammankoppla två affibodybaserade VEGFR2- bindare, Zdimer och Ztetramer, till 4RepCT. Bibehållen aktivitet av de kopplade affibody-molekylerna bekräftades genom receptorfosforylering och cellproliferationsstudier. Dessutom rapporterades möjligheten att skapa

iii kärlliknande strukturer inuti ett FN-silkesbaserat cell-scaffold innehållande Ztetramer- silkesfibriller. Dessa resultat lyfter fram den framtida potentialen hos de utvecklade silkesbaserade materialen för regenerativ medicin.

List of publications

This thesis is based on the work contained in the following papers, referred to by Roman numerals in the text:

I Jansson, R., Thatikonda, N., Lindberg, D., Rising, A., Johansson, J., Nygren, PÅ., Hedhammar, M. Recombinant spider silk genetically functionalized with affinity domains. Biomacromolecules (2014), 15(5), 1696-1706.

II Thatikonda, N., Delfani, P., Jansson, R., Petersson, L., Lindberg, D., Wingren, C., Hedhammar, M. Genetic fusion of single-chain variable fragments to partial spider silk improves target detection in micro- and nanoarrays. Biotechnology Journal (2016), 11(3), 437-448.

III Thatikonda, N., Nilebäck, L., Lundin, A., Widhe, M., Hedhammar, M. Bioactivation of spider silk with basic fibroblast growth factor for in vitro cell culture: a step towards creation of artificial ECM. Biomaterials Science & Engineering (2018), 4(9), 3384–3396.

IV Güler, R.*, Thatikonda, N.*, Hawraa, Ali Ghani., Hedhammar, M., Löfblom J. Artificial VEGFR2-Specific Growth Factors Demonstrate Agonistic Effects in Both Soluble Form and When Immobilized Via Spider Silk. Manuscript.

* These authors contributed equally

Paper I-IV are reproduced with the permission from the copyright holders.

Respondent’s contributions to the included publications

I Planned the studies, performed laboratory work and analyzed data for the work related to the fourth construct. Participated in the discussions related to other constructs together with minor contribution to writing the manuscript.

II Planned the studies, performed majority of the work related to the construction, expression and purification of all recombinant silk constructs employed in the study. Analyzed results together with major contribution to writing the manuscript.

III Planned the studies, performed majority of the work reported in the study, except AFM imaging and calculation of viscoelastic properties using QCM-D. Analyzed results together with major contribution to writing the manuscript.

IV Planned the studies, performed majority of the work related to Zdimer and Ztetramer domains immobilized to silk. Analyzed results and contributed to writing the manuscript.

Published work not included in the thesis

Chouhan, D., Thatikonda, N., Nilebäck, L., Widhe, M., Hedhammar, M., Mandal, BB. Recombinant spider silk functionalized silkworm silk matrices as potential bioactive wound dressings and skin grafts. Applied Materials and Interface (2018) 10, 23560−23572.

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Public defense of dissertation This thesis will be defended on October 31st, 2018 at 10:15, Oskar Kleins Auditorium, Roslagstullsbacken 21, AlbaNova University Center, Stockholm, for the degree of “Teknologie Doktor” (Doctor of Philosophy, PhD) in Biotechnology.

Respondent: Naresh Thatikonda, M.Sc. in Biology Department of Protein Science, School of Engineering Sciences in Chemistry, Biotechnology and Health KTH Royal Institute of Technology, Stockholm, Sweden

Faculty opponent: Hanna Dams-Kozłowska PhD (Dr hab. n. med.) Head of Cancer Immunology Lab, Greater Poland Caner Center, Poznan, Poland

Evaluation committee: Assoc. Prof. Natalia Ferraz Department of Engineering Sciences, Nano Technology and Functional Materials, Uppsala University, Uppsala, Sweden

Assoc. Prof. Liya Wang Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, SLU, Uppsala, Sweden

Assoc. Prof. Jakob Dogan Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden

Chariman: Prof. Torbjörn Gräslund Department of Protein Science, School of Engineering Sciences in Chemistry, Biotechnology and Health KTH Royal Institute of Technology, Stockholm, Sweden

Respondent’s main supervisor: Assoc. Prof. My Hedhammar Department of Protein Science, School of Engineering Sciences in Chemistry, Biotechnology and Health KTH Royal Institute of Technology, Stockholm, Sweden

Respondent’s co-supervisor: Assoc. Prof. Christofer Lendel Division of Applied Physical Chemistry, Department of Chemistry, School of Engineering Sciences in Chemistry, Biotechnology and Health, Stockholm, Sweden

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Abbreviations

ABD Albumin binding domain B. mori Bombyx mori bFGF Basic fibroblast growth factor CT C-terminal domain of spidroin E. australis Euprosthenops australis E. coli Escherichia coli EGF Epidermal Growth Factor ECM Extracellular Matrix Fab Fragment antigen-binding Fc Fragment crystallizable FTIR Fourier transform infrared spectroscopy IMAC Immobilized Metal ion Affinity Chromatography IgG Immunoglobulin MaSp Major ampullate spidroin M4 Monomeric streptavidin NT N-terminal domain of spidroin N. clavipes Nephila clavipes NTA Nitrilotriacetic acid scFv Single chain variable fragment SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis SPA Staphylococcal protein A SPG Streptococcal protein G VEGF Vascular Endothelial Growth Factor

To my family and friends

May all be happy; May all be without disease; May all have well-being; May none have misery of any sort. -Brihadaranyaka Upanishad

All of us do not have equal talent, but all of us have an equal opportunity to develop our talents. -Dr. APJ Abdul Kalam

Contents

Abstract i Abstract in Swedish iii List of publications v Public defense of dissertation viii Abbreviations ix

1 Introduction 1 1.1 Natural spider silk 1 1.2 Overview of recombinant spider silk 3 1.2.1 Recombinant production of partial spider silk in bacteria 3 1.2.2 Processing of partial spider silk proteins to various formats 5 1.2.3 Modification of partial spider silk proteins 5 1.3 Different ways to immobilize proteins onto solid supports 5 1.4 Affinity domains 7 1.4.1 Domains derived from bacterial surface receptors 7 1.4.2 Domain derived from streptavidin 8 1.4.3 Domains derived from antibodies 8 1.5 Diverse applications of functionalized partial spider silk 9 1.6 Overview of protein microarrays 10 1.6.1 Recombinant antibody fragment microarrays 12 1.6.2 Key parameters in affinity domain based microarrays 13 1.6.3 Applicability of different affinity domains for microarrays 14 1.6.4 Immobilization of affinity domains for sensitive recombinant antibody microarrays 14 1.7 Essential components of the cellular microenvironment 15 1.7.1 Neighboring cells 15 1.7.2 Physical fields 16 1.7.3 Extracellular matrix 16 1.7.4 Soluble factors 17 1.8 Overview of tissue engineering 20 1.8.1 Rationale of tissue engineering 22 1.8.2 Characteristics of a biomaterial for its use in tissue engineering 23 1.8.3 Biomaterials in tissue engineering 24 1.8.4 Role of growth factors in tissue engineering 25

2 Present investigations 29 2.1 Aims of the thesis 29

2.2 Results and discussion 30 2.2.1 Paper I: Recombinant spider silk genetically functionalized with affinity domains. 30 2.2.2 Paper II: Genetic fusion of single-chain variable fragments to partial spider silk improves target detection in micro- and nanoarrays. 35 2.2.3 Paper III: Bioactivation of Spider Silk with Basic Fibroblast Growth Factor for in vitro Cell Culture: A Step toward Creation of Artificial ECM. 40 2.2.4 Paper IV: Artificial VEGFR2-Specific Growth Factors Demonstrate Agonistic Effects in Both Soluble Form and When Immobilized Via Spider Silk. 47

3 Concluding remarks and future perspectives 52

4 Popular scientific summary 55

Acknowledgements 57

References 60

Sections 1.1-1.5, 2.2.1-2.2.2 and 4 are modified from respondent’s licentiate thesis.

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1 Introduction

The capability to generate silk, a natural protein fiber, has been developed by spiders, several arthropods and 23 groups of insects (Walker et al., 2012; Sutherland et al., 2010; Altman et al., 2003). Due to the remarkable mechanical and elastic properties of the silk derived from spiders, combined with its biocompatibility, spider silk has been exploited for biomaterial and biomedical applications (Borkner et al., 2014; Bittencourt et al., 2012; Widhe et al., 2012; Omenetto & Kaplan, 2010). However, additional functionalization is needed for generation of more advanced materials, which otherwise does not exist in nature. Processing of silk from cocoons produced by silkworms require harsh processing steps involving for example degumming in alkali solution followed by solubilization in organic solvents like hexafluoroisopropanol, which restrict their use in applications where additional functionalization is needed (Rockwood et al., 2011). The work described in this thesis is mainly based on the silk derived from spiders, thus information regarding other kinds of silk is not described, but can be found elsewhere (Kasoju & Bora, 2012; Sutherland et al., 2010).

1.1 Natural spider silk In nature, orb weaving spiders can make seven different silks, which have different physical properties for different functions (Rising et al., 2011). Out of the seven types of spider silks, dragline silk that is produced in the major ampullate gland of spider has intensely been studied by researchers for its incredible mechanical properties (Borkner et al., 2014; An et al., 2012; Bittencourt et al., 2012). Spiders use dragline silk as a lifeline to hang safely from their webs, and also to make frames of their webs. A movie scene

performed by superhero from Spider-Man 2, released in 2004, where a subway train is stopped by his webbing is not far from the reality. Researchers from University of Leicester, UK are in the process to fabricate synthetic spider silk fibers with extraordinary toughness (Bourzac, 2015). Spider dragline silk is composed of two high molecular weight proteins (Ayoub et al., 2007; Bini et al., 2006), namely Major ampullate spidroin 1 and 2 (MaSp1 and MaSp2, respectively) (Humenik et al., 2011). The schematics of spider major ampullate silk is illustrated in Figure 1. The dragline silk spidroins produced by various species of spiders share a common tripartite protein architecture (Humenik et al., 2011). They are composed of a non-repetitive N- terminal domain, a repetitive region mainly containing poly-alanine segments interspersed between glycine rich repeats, and a non-repetitive C-terminal domain (Ayoub et al., 2007). The repetitive region, which is composed of different amino acid segments, was reported to account for the amazing physical properties of the dragline silk (Rising et al., 2011). Naturally, spiders produce silk protein in liquid form (dope) and store it at high concentrations (50% w/v) within specialized silk glands (Chen et al., 2002). Upon changes in the ion composition, pH, dehydration (Walker et al., 2012), and shear forces occurring along the duct (Dicko et al., 2004; Knight & Vollrath, 2001); stored silk solution will emerge out as silk fibers, which are rich in β-sheets (Gosline et al., 1999; Simmons et al., 1996). The intermolecular interactions occurring between the poly-alanine segments and glycine rich repeats in the repeat region (Rising et al., 2011), combined with the N-terminal (Askarieh et al., 2010) and C-terminal domain (Hagn et al., 2010) of the spidroins were reported to regulate the process of silk fiber formation. The dragline silk of Euprosthenops is stronger than that from other species of spiders like Nephila, Araneus and Latrodectus (Stark et al., 2007; Pouchkina-Stantcheva & McQueen-Mason, 2004). Advantages and limitations in production of natural and engineered dragline silks are addressed in a later section.

Figure 1. Schematics of the repetitive region of spider major ampullate (MA) silk. The silk fiber is composed of crystalline rich regions covalently joined by noncrystalline regions. Strength and elasticity are provided by the crystalline and noncrystalline regions, respectively. Inspired from (Romer & Scheibel, 2008).

1.2 Overview of recombinant spider silk

1.2.1 Recombinant production of partial spider silk in bacteria The predatory behavior of spiders together with the insufficient amounts of silk produced by spiders, have restricted the option to perform sericulture of spiders and hence opted for recombinant production of spider silk proteins (Guhrs et al., 2000). Recombinant production is defined as the expression of a foreign gene in a production host, for example bacteria (Tokareva et al., 2013). Due to the well characterized genome and low costs involved in production, Escherichia coli (E.coli) is often considered as a suitable option for large scale protein production. Due to the repetitive nature and large size of natural spider silk genes in general, issues like premature translational stops, low protein yield and homologous recombination to host genome, can be of concern when expressed in the bacteria (Arcidiacono et al., 1998). For this reason, partial spider silk sequences are preferred for production of artificial spider silk proteins in bacteria (Tokareva et al., 2013; Stark et al., 2007; Prince et al., 1995). With recent progress in the field of genetic engineering together with the modular

nature of spider silk, different partial spider silk proteins derived from different species of spiders have been produced in various hosts including yeast, bacteria, insects, plants and mammalian cells with varying success (Tokareva et al., 2013; Rising et al., 2011). Some of the partial spider silks which were cloned and expressed in bacteria include repeat segments derived from MaSp1 and MaSp2 of Nephila clavipes with (Prince et al., 1995) or without (Mello et al., 2004) inclusion of a native C-terminal domain. Another example of partial spider silk is 4RepCT, composed of four poly-alanine/glycine rich co-segments together with a C-terminal domain derived from MaSp1 of Euprosthenops australis (E. australis) (Stark et al., 2007). Likewise, another partial spider silk variant, NTCT was herein generated by combining native N-terminal and C- terminal domains derived from E. australis. The study presented in this thesis is based on these two partial spider silk variants, 4RepCT and NTCT. The major steps involved in the recombinant production of partial spider silk proteins in bacteria are (Figure 2): (i) Isolation of full length spider silk cDNA from spider, (ii) Selection of cDNA that codes for partial spider silk protein, (iii) restriction cleavage of target vector, (iv) ligation of partial spider silk cDNA to the cleaved target vector, (v) transformation of generated recombinant DNA (rDNA) into bacteria, (vi) expression of rDNA in the bacteria, (vii) harvesting and purification of the partial spider silk proteins from the bacteria, mainly using affinity purification (Stark et al., 2007; Prince et al., 1995). All the steps are represented in Figure 2.

Figure 2. Illustration of recombinant production of partial spider silk proteins in bacteria. (i) Isolation of full length cDNA libraries from spider, (ii) selection of cDNA libraries that

codes for partial spider silk, (iii) cleavage of target vector, (iv) ligation of selected cDNA of partial spider silk into cleaved target vector, (v) transformation of rDNA to bacteria, (vi) Recombinant protein expression in bacteria, (vii) purification of partial spider silk protein

1.2.2 Processing of partial spider silk proteins to various formats The success of recombinant production in bacteria has enabled a convenient way to produce partial spider silk proteins under denaturing (Humenik et al., 2011) and non-denaturing conditions (Stark et al., 2007). Moreover, the produced partial spider silk proteins have an advantage of being possible to process into unnatural silk morphologies like, foam, film and mesh formats with (Hardy & Scheibel, 2009) or without (Widhe et al., 2010) being subjected to post-treatment with organic solvents for β-sheet enrichment. Partial spider silk variants which are produced under non-denaturing conditions without any requirement of organic solvents during material fabrication might have better opportunities for modification with a wide range of biologically active molecules, e.g. affinity and bioactive domains.

1.2.3 Modification of partial spider silk proteins Chemical or genetic modification of partial spider silk proteins will broaden the applicability window of silk based materials (Wohlrab et al., 2012; Spieß et al., 2010). Chemical modification can be achieved by the use of established procedures that permit efficient and site-specific modifications of proteins (Hackenberger & Schwarzer, 2008). However, certain coupling chemicals used for modification of silk materials might cause side-effects that are detrimental to cells when used for tissue engineering applications (Wohlrab et al., 2012). In those cases, genetic modification of partial spider silk proteins could be used as a more beneficial alternative. The possibility to genetically modify partial spider silk has been shown by the addition of cell binding motifs using genetic engineering method with convincing results in cell culture studies (Widhe et al., 2016; Widhe et al., 2013; Wohlrab et al., 2012).

1.3 Different ways to immobilize proteins onto solid supports Methods which rely on the binding of target molecules to complementary “bait” molecules depend on the immobilization of those bait molecules to a solid support. This section describes different ways to immobilize proteins, which constitute one important group of such bait molecules. For example,

antibodies are immobilized to solid support for their use in affinity purification and immunoassays. Immobilization of proteins on solid surfaces is of great importance in the production of protein based arrays and biosensor related applications. The challenging task is to immobilize the proteins in such way that their functionality and orientation are maintained, and also to remain their conformation unaltered after immobilization (Oshige et al., 2013). Furthermore, restricting the interaction between the immobilized antibodies and the solid surface might help to achieve an appreciable portion of antibodies that retain their native confirmation. Addressing the above challenges can allow for the successful use of immobilized proteins for their intended applications. The chemical and structural complexity of proteins have created difficulties in determining a general strategy for immobilization of proteins (Saerens et al., 2008). Different ways to immobilize proteins on various surfaces have been reported, namely 1) non-covalent physical adsorption, 2) covalent coupling via free amines or thiol groups and 3) non-covalent protein linkage via affinity interactions (e.g.

His6/Ni-NTA) (Oshige et al., 2013; Kimple et al., 2010). In all cases, physical and chemical properties of the surface and also other factors like temperature and pH have been shown to affect the surface immobilization of the proteins (Oshige et al., 2013). Non-specific physical adsorption is a quick way to immobilize protein molecules, but this often leads to protein denaturation (Butler et al., 1993). In order to prevent the denaturation of immobilized proteins, they can be spotted onto solid surfaces, which have been pre-coated with e.g. polyacrylamide gels or polyethylene glycol. The hydrophilic environment provided by polyacrylamide gels was reported to protect against shear deactivation of immobilized proteins (Pollak et al., 1980). In the covalent coupling method, reactive functional groups present in the protein molecules (e.g., amines, thiols) are crosslinked onto the solid support modified with complementary reactive groups, thus ensuring protein immobilization (Oshige et al., 2013; Camarero, 2008). However, the immobilization methods described above does often not result in well oriented protein molecules, which is necessary for efficient binding of their target molecules (Oshige et al., 2013; Camarero, 2008). The orientation of immobilized protein molecules can be improved by use of a non- covalent protein immobilization via affinity. For example, proteins carrying histidine tags can be immobilized onto solid surface with Ni-NTA (Nickel-

Nitrilotriacetic acid). However, non-covalent means of immobilization is limited by the lack of long-term stability of the immobilized proteins (Camarero, 2008).

1.4 Affinity domains One important class of proteins that is beneficial to have immobilized onto surfaces for certain applications e.g. in vitro diagnosis, are affinity domains.

1.4.1 Domains derived from bacterial surface receptors In the recent past, exploiting the host defense mechanisms developed by bacteria for their use in the field of biotechnology has gained considerable attention (Diamandis & Christopoulos, 1991). Bacterial strains of the Staphylococci, Streptococci and Streptomyces genera express certain proteins in order to evade the host immune system (Sauer-Eriksson et al., 1995; Diamandis & Christopoulos, 1991). Out of the several IgG-binding bacterial proteins that were identified, staphylococcal protein A (SPA) and streptococcal protein G (SPG) are extensively studied for their use in the purification of antibodies (Boström et al., 2012). SPA contains five homologous IgG-binding domains. The Z domain (7 kDa) (Figure 3), which folds into a three-helix bundle, is an engineered protein domain derived from the IgG-binding B-domain of SPA (58 kDa) (Boström et al., 2012; Nilsson et al., 1987). SPG has a multi domain structure containing both albumin-binding domains and immunoglobulin-binding domains (Kraulis et al., 1996). The C2 domain (6 kDa) (Figure 3), which folds into four β-sheets and α-helix structure is derived from the Fc binding domain B1 of SPG (Sauer- Eriksson et al., 1995). Both Z and C2 domains can be used for the purification of antibodies or target proteins fused to the Fc domain. SPA and SPG exhibit different specificities to IgG obtained from different species (Boström et al., 2012). Likewise, the domains derived from SPA and SPG could possibly exhibit species specific IgG binding. The ABD domain is an engineered protein domain derived from albumin- binding domain 3 of SPG (63 kDa) (Figure 3). Domain ABD (5 kDa) has affinity towards human serum albumin and folds into a three-helix bundle (Nilvebrant & Hober, 2013; Kraulis et al., 1996).

Figure 3. Ribbon structure representations of the Z domain (a) (PDB file: 2SPZ (Tashiro et al., 1997)), the C2 domain (b) (PDB file: 1FCC (Sauer-Eriksson et al., 1995)) and the ABD domain (c) (PDB file:1GJT (Johansson et al., 2002)).

1.4.2 Domain derived from streptavidin Streptavidin is a protein produced by Streptomyces avidinii, which has affinity towards biotin and biotinylated ligands (Diamandis & Christopoulos, 1991). The extremely high affinity (fM range) between biotin and streptavidin (Diamandis & Christopoulos, 1991) complicate the release of biotinylated ligands (Rybak et al., 2004). This resulted in the generation of monomeric streptavidin (M4) (Wu & Wong, 2005), which exhibit a lower binding affinity than streptavidin, in order to expand the applicability of biotin/streptavidin system for several biotechnological applications. M4 is a 16.5 kDa monomeric version of streptavidin, which has affinity to biotin molecules and folds into a β-sheet barrel structure (Wu & Wong, 2005).

1.4.3 Domains derived from antibodies Naturally, antibodies are produced by B-cells in order to protect against foreign substances. As represented in Figure 4, antibodies are composed of two heavy and two light chains joined together by disulfide bonds. There are three constant domains and one variable domain in the heavy chains, while there is one constant domain and one variable domain in the light chains (Figure 4). The epitope recognition site of an antibody is in the variable heavy (VH) and variable light (VL) domains. Due to excellent target specificity and affinity, antibodies are considered as paradigm for cell biology and biochemistry applications (Gebauer & Skerra, 2009). However, structural complexity and laborious procedures involved in the production of full-length antibodies have led to creation of recombinant antibody fragments such as scFv and Fab (Gebauer & Skerra, 2009) (Figure 4). Considerable loss of biological activity has been observed upon immobilization of antibodies onto solid supports, which affects the sensitivity of the antibody based immunoassays, for example. Furthermore, the presence of the Fc part of the antibody can result in unwanted

cell reactivity upon binding to the effector cells containing Fc receptors (Holliger & Hudson, 2005). Generation of antibody mimics was considered as a breakthrough in the field of antibody engineering. Monovalent Fabs (55 kDa) are generated from full-length antibodies subjected to papain cleavage (Porter, 1959), whereas, single-chain variable fragment, scFv (28 kDa), one of the most popular formats of antibody derivatives, is generated by the fusion of variable domains of the heavy and light chain (VH and VL) joined by a flexible linker. Generated Fab and scFv fragments are smaller in size when compared to the antibodies (Figure 4) and can be expressed in E. coli with retained antigen binding affinity of the intact IgG (Bird et al., 1988). Due to the smaller size of scFv and due to less efficient folding and assembly of Fab fragments in E. coli (Hust et al., 2007), scFv is considered as a suitable affinity ligand for different applications, immunoassays for example. The biomolecular properties, like stability, can be enhanced by engineering scFvs (Worn & Pluckthun, 2001).

Figure 4. Illustration of an antibody and domains derived from an antibody.

1.5 Diverse applications of functionalized partial spider silk Development of functionalized partial spider silk for diverse applications is an actively growing field of research. Partial spider silk proteins, which can be produced using recombinant DNA technology, can be subjected to genetic modifications by incorporation of peptides, recognition motifs or functional domains (Borkner et al., 2014). In the recent past, cell binding peptides have successfully been incorporated to different engineered spider silk proteins, thereby suggesting the applicability of such functional silk materials for in vitro cell culture studies (Widhe et al., 2013; Wohlrab et al., 2012). Furthermore,

silver binding peptides (Currie et al., 2011) and antimicrobial peptides (Gomes et al., 2011) were genetically incorporated to partial spider silk and the added peptides were reported to retain their functionalities, thus highlighting the use of silk-silver and silk-antimicrobial materials for biomedical applications. Likewise, uranium recognition motifs were genetically fused to a partial spider silk protein derived from N. clavapies, and then the uranium binding ability of the chimeric silk-uranyl proteins were confirmed. This highlights the applicability of such functional silk fusion proteins as biosensor capture probe for environmental monitoring applications (Krishnaji & Kaplan, 2013). Affinity domains are complex and larger in size when compared to the peptides or recognition motifs. Therefore, exploring the possibility to incorporate affinity domains to partial spider silk might increase the applicability of functional silk based materials for other applications, for example immunoassays, advanced biosensor and cell culture applications.

1.6 Overview of protein microarrays The fundamental principle of microarray assays was first described by Roger Ekins in 1980 (Ekins, 1989). The protein microarray is a versatile system that can be used for characterization of thousands of proteins on a small slide in a high-throughput manner. For this reason the microarray technology is considered as miniaturization of several assays that can be performed on a small slide (Reymond Sutandy et al., 2013). Based on the mode of their applications, protein microarrays are generally classified into analytical (affinity protein microarrays) and functional protein microarrays (Reymond Sutandy et al., 2013; Zhu & Qian, 2012; Wingren & Borrebaeck, 2006). In functional protein microarrays, considerable number of individually purified proteins are spotted onto an array slide mainly to study the biochemical interactions of the immobilized proteins. Therefore, functional protein microarrays can mainly be used to study protein-protein, protein-DNA and protein-RNA interactions, for example (Zhu & Qian, 2012). Whereas in affinity protein microarrays, well characterized capture molecules, e.g. antibodies, are used to validate the presence of an analyte of interest for example a disease biomarker in a non-fractionated sample like serum (Zhu & Qian, 2012; Wingren & Borrebaeck, 2006). Thus, affinity protein microarrays can be used for disease diagnosis and discovery of novel biomarkers, for example (Kumble, 2003). An illustration of a standard affinity protein

microarray is represented in Figure 5, where the analyte of interest can be detected by direct labeling or using a sandwich approach via a reporter antibody.

Figure 5. Illustration of a general affinity protein microarray. Directly labeled analyte: for analyte detection by direct labeling; Sandwich: for analyte detection using a reporter antibody. One capture molecule shown in the illustration is representative of several capture molecules present per spot.

In the next sections, the use of recombinant antibodies as capture molecules for affinity protein microarrays is first described. Thereafter, a general procedure to perform recombinant antibody arrays is explained, followed by a brief description of key technological limitations involved in generation of high performing microarrays. While discussing key technological limitations, major focus is devoted towards describing different strategies used for immobilization of affinity domains, e.g. recombinant antibodies, onto solid supports for their efficient use in microarrays.

1.6.1 Recombinant antibody fragment microarrays For several years, full-length antibodies have been used as capture probes in protein microarrays. This is due to the high specificity and affinity exhibited by antibodies towards their target molecules (Zeng et al., 2012; Borrebaeck & Wingren, 2011; Hartmann et al., 2009). However, the production and validation of antibodies is time-consuming and expensive as well (Hartmann et al., 2009). In order to reduce the costs related to antibody assays, researchers have used genetic engineering techniques for creation of other capture probes, such as antibody fragments (as opposed to full-length antibodies) for their use in microarrays (Zeng et al., 2012; Borrebaeck & Wingren, 2011). Over the years, various formats of recombinant antibodies have been designed, including single-chain fragments variable (scFv), Fab fragments, diabodies and nanobodies (single-domain antibody) (Holliger & Hudson, 2005). Additionally, genes encoding recombinant antibodies can be introduced in bacteria, yeast and other cell lines, thus making them suitable for animal-free and cost-effective production (Hu et al., 2013; Marks et al., 1991). Recombinant antibody microarrays can be used to detect protein biomarkers and levels thereof, which are specific to a disease, from a complex sample like human blood serum, for example (Hartmann et al., 2009). According to the National Institutes of Health (NIH) Biomarkers definitions working group, a biomarker is defined as “A characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention” (Strimbu & Tavel, 2010). In recombinant antibody microarrays, hundreds of antibody fragments that can simultaneously detect different analytes (e.g. disease biomarkers) are attached by either direct adsorption or covalent immobilization onto a microarray slide (an area of <1 cm2). Illustration of general recombinant antibody array is represented in the Figure 6. The steps involved in detection of an analyte of interest includes: (i) Selection of microarray slide with appropriate surface chemistry followed by printing of recombinant antibodies onto the array slide. (ii) Biotin labelling of a serum sample containing protein biomarkers followed by their addition onto the microarray slide. (iii) Addition of fluorophore labelled streptavidin for the detection of bound biotinylated protein biomarkers that are bound to the recombinant antibodies. (iv) Scanning of the array slide to obtain a protein signature for a particular disease by following a series of steps, including signal quantification, data pre-processing, data analysis thereby generating a protein expression profile from which a

protein signature for a particular disease can be obtained. (Borrebaeck & Wingren, 2009).

Figure 6. General illustration of the different steps required for protein detection using a protein microarray. (i) Spotting or printing of recombinant antibodies that detect disease biomarkers. (ii) Biotin labeling of a non-fractionated serum sample containing disease biomarkers followed by its addition onto the microarray slide. (iii) Addition of fluorophore labeled streptavidin onto the microarray slide for the detection of bound biotinylated disease biomarkers. (iv) Scanning of the array slide to obtain an immunosignature.

1.6.2 Key parameters in affinity domain based microarrays In protein-based detection methods, appropriate immobilization of capture molecules such as antibodies and affinity domains, e.g. antibody fragments, are considered as an important aspect in creation of stable capture probes (Shen et al., 2017), thereby contributing to improvement in sensitivity of the detection method. There exists considerable advantages with enhanced sensitivity, such as possibility to achieve highly accurate molecular profiling and to be able to detect novel, low-abundant protein biomarkers. Enhanced sensitivity is an important feature to be considered for creation of efficient ligand-analyte detection systems. Stability, selectivity and orientation of recombinant antibodies spotted on the array slide represent one of the important technological parameters to consider for the design of high performing recombinant antibody microarrays (Shen et al., 2017). Detailed description of

other technological parameters is out of the scope of this thesis and are described elsewhere (Wingren & Borrebaeck, 2006).

1.6.3 Applicability of different affinity domains for microarrays In the recent times, various types of affinity reagents have been used as capture probes in microarrays, including antibodies, recombinant antibodies (e.g. scFv, Fab) (Holliger & Hudson, 2005), non-immunoglobulin-derived affinity reagents (e.g. affibodies) (Nygren, 2008) and oligonucleotide apatamers (Ellington & Szostak, 1990). Target binding studies of the recombinant antibodies and affibodies hinted that they recognize different surface architectures present on the target antigen (Affinomics, 2013). This suggests that the use of different affinity domains towards the same target is a choice, which cannot be ignored. This information could be beneficial for selection of appropriate affinity domains as capture probes in microarrays.

1.6.4 Immobilization of affinity domains for sensitive recombinant antibody microarrays The method used for immobilization of affinity domains is important to consider for generation of sensitive antibody arrays. Upon use of a certain immobilization technique, it is crucial that the immobilized affinity domains remain stable, maintain their structure, function and have access to their target binding sites. Thereby, the ability of the immobilized affinity domain (ligand) to detect its specific proteins in a patient sample (analyte) can be improved, thus, increasing the overall sensitivity of the microarray assay. The considerable variations in physiochemical properties of different affinity domains could possibly be the reason for lack of a universal protein immobilization method. Different ways to immobilize proteins onto solid supports is previously described in 1.3 section in this thesis. Similar strategies can be used for immobilization of affinity domains as well, with the aim to achieve stable capture probes for generation of sensitive microarrays. In Paper II, a novel strategy for immobilization of antibody fragment (scFv) onto partial spider silk proteins (4RepCT and NTCT) via genetic engineering is described. Furthermore, the applicability of scFv-silk fusion proteins for creation of sensitive arrays is investigated.

1.7 Essential components of the cellular microenvironment Development of people in the society is dependent on their local environment. Likewise, cell growth and development are strongly dependent on its microenvironment. Cells in vivo reside surrounded by a complex, heterotypic and dynamic set of biochemical and biophysical cues, which make out the “cellular microenvironment”. The cellular microenvironment provides structural support to the residing cells and regulate cell behavior (Huang et al., 2017). Generally, the cellular microenvironment contains four essential components, including neighboring cells, physical fields, extracellular matrix (ECM) and soluble factors (Figure 7). These four key components act synergistically to influence cell outcomes, e.g. adhesion, proliferation, migration, differentiation (Huang et al., 2017). A brief description of neighboring cells and physical fields are presented in the first part of this section. Then, special attention is devoted towards extracellular matrix and soluble factors, in particular growth factors and their signaling.

1.7.1 Neighboring cells In vivo, cells communicate with similar and diverse types of cells. Several studies have determined the important role of cell-cell interactions in regulating cell behavior and tissue development (McMillen & Holley, 2015). Normally,

cell-cell adhesion is governed by specific adhesion receptors such as cadherins, whereas, cell-ECM adhesion is dominated by integrins (Li et al., 2017; McMillen & Holley, 2015). Briefly, cell-cell interaction can occur by two mechanisms, namely 1) by direct cell-cell contact (via tight junctions, anchor junctions and gap junctions) and 2) by soluble factors (via growth factors) (Huang et al., 2017).

1.7.2 Physical fields In addition to the cell-cell interactions described in the above section, physical fields are also considered as important components of the cellular microenvironment (Figure 7). Depending on the location in vivo the cells are influenced by several physical fields, such as strain and stress (Huang et al., 2017). Cells sense these physical fields by cell receptors and cytoskeleton via a process called mechanosensing, which activates intracellular signaling pathways (mechanotransduction), thereby regulating the cell responses (Luo et al., 2013; Vogel & Sheetz, 2006).

1.7.3 Extracellular matrix Every cell in human tissues is surrounded by a complex three dimensional (3D) fibrillar network named the extracellular matrix (ECM). This is one of the essential components of the cellular microenvironment (Figure 7). The ECM is an intricate and dynamic network, which provides biophysical, biochemical and mechanical signals to the residing cells (Gattazzo et al., 2014). The ECM is composed of a network of various macromolecules like collagen, proteoglycans, glycosaminoglycans, fibronectin, laminins, growth factors and other signaling molecules, which can modulate cell behavior (Lee et al., 2011) (Figure 8). Briefly, hyaluronic acid, dermatan sulfate, keratan sulfate, chondroitin sulfate and heparin sulfate belongs to the group of sulfated glycosaminoglycans (GAGs). GAGs are chemically conjugated to a protein filament to form proteoglycans (Figure 8). The main functions of proteoglycans are to regulate the hydration level of the ECM and to capture and maintain activity of soluble molecules (e.g. growth factors) secreted by the cells (Carmagnola et al., 2018; Patterson et al., 2010). ECM based cell regulation plays a crucial role during development and can either be achieved by direct or indirect mechanisms (Hynes, 2009). ECM proteins that contribute to direct modulation of cell behavior includes structural proteins (collagen and elastin) and cell adhesion proteins (e.g. fibronectin,

laminin, vitronectin) that interacts with cell surface receptors (mainly integrins) (Carmagnola et al., 2018). Indirect modulation of cell regulation can be achieved by growth factors, which are captured due to their affinity towards ECM components, such as proteoglycans (Figure 8). Thus, ECM bound growth factors contribute to directing cell migration, differentiation and maturation (Patterson et al., 2010; Shastri & Lendlein, 2009). In the recent years, considerable focus is kept on the creation of materials that can mimic the structures and functions of native ECM. Thereby, in vitro study of cells in a realistic cellular microenvironment can be achieved (Huang et al., 2017; Murphy et al., 2014; Wade & Burdick, 2012). As a step toward mimicking native ECM, the possibility to co-present cell adhesion ligands and growth factors in a spider silk scaffold is investigated in Paper III presented in this thesis.

Figure 8. Schematic illustration of the major components of ECM and cell-ECM interactions. Figure inspired from https://www.khanacademy.org/science/biology/structure- of-a-cell/cytoskeleton-junctions-and-extracellular-structures/a/the-extracellular-matrix-and- cell-wall

1.7.4 Soluble factors As illustrated in Figure 7, growth factors are among other soluble factors (e.g. amino acids, oxygen, glucose, cytokines) the important molecules that cells

encounter in vivo, and they are considered essential for creation and maintenance of the cellular microenvironment (Huang et al., 2017). Albeit growth factors are available only in small quantities within ECM, yet they act as effective regulators of cell response and are widely used for engineering of the biomimetic cellular microenvironment (Huang et al., 2017; Netti, 2014; Badylak et al., 2008).

1.7.4.1 Growth factors Growth factors are essential signaling molecules secreted in soluble form. They provide instructions to cells during the developmental stage, and may promote tissue regeneration during adult stage (Lee et al., 2011). Growth factors can act in four different ways; either act on the same cell as it was secreted from (autocrine mode), secreted by neighboring cells (paracrine mode), or distributed by the circulatory system (endocrine mode). In certain instances, growth factors exert their cell bioactivity in non-diffusible manner (juxtacrine mode) (Huang et al., 2017; Hajimiri et al., 2015). A list of selected growth factors and their physiological functions are stated in Table 1.

Table 1. List of selected growth factors and their biological functions. Growth factor Biological function EGF Regulation of epithelial cell growth, proliferation and differentiation bFGF Survival, proliferation and migration of endothelial cells, maintain (or FGF-2) pluripotency of embryonic stem cells by inhibiting their differentiation BMP-2 Differentiation and migration of osteoblasts BMP-7 Differentiation and migration of osteoblasts, renal development NGF Survival and proliferation of neural cells PDGF-AB Embryonic development, proliferation, migration, growth of endothelial (or-BB) cells VEGF Survival, proliferation and migration of endothelial cells. EGF, epidermal growth factor; bFGF, basic fibroblast growth factor, BMP, bone morphogenetic protein; NGF, nerve growth factor; PDGF, platelet-derived growth factor; VEGF, vascular endothelial growth factor. Table is modified from (Lee et al., 2011)

The in vivo cell environment contains both soluble and immobilized growth factors with distinctive functions (Figure 8). Thus, the experimental immobilization of growth factors in vitro does have physiological relevance (Hajimiri et al., 2015). Furthermore, upon immobilization the amount of growth factor required is reduced. Thereby, the cost involved in growth factor

delivery systems can be decreased (Hajimiri et al., 2015). For the reasons specified above, the design of biomaterials that can interact with growth factors are compelling. Strategies used for immobilization of protein domains are discussed in an earlier part of this thesis (Section 1.3). Similar conjugation strategies can be used for growth factor immobilization onto various surfaces. Detailed description of popular growth factors as listed in Table.1, and their conjugation strategies, are further described elsewhere (Hajimiri et al., 2015; Tallawi et al., 2015; Lee et al., 2011).

1.7.4.2 Receptor Tyrosine Kinase/growth factor signaling Cells communicate with their microenvironment via cell membrane receptors e.g. integrins and growth factor receptors (Figure 8). The functions of growth factors are thus regulated by the growth factor receptors. Receptor Tyrosine Kinases (RTKs) function as receptors for several types of growth factors, including epidermal growth factor (EGF), platelet-derived growth factor (PDGF), nerve growth factor (NGF), fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) (Popovic & Wilson, 2018). As illustrated in Figure 9, the RTKs are transmembrane protein receptors containing an extracellular ligand binding domain, a transmembrane part and the cytoplasmic part containing tyrosine kinase domain (TKD) (Lemmon & Schlessinger, 2010; Ullrich & Schlessinger, 1990). Binding of a ligand (growth factor) to the extracellular domain (RTK) result in activation of the intracellular kinase domain with subsequent auto- phosphorylation and phosphorylation of various intracellular substrates that are involved in the signal transduction process (Schlessinger, 1988). This results in transfer of signal to the nucleus, so that genes essential for cell survival, growth, proliferation or differentiation will be transcribed (Figure 9). Signaling pathways activated by RTKs include: Mitogen-activated protein kinase (MAPK), extracellular-signal-regulated kinase (ERK), and phosphatidylinositol 3-kinase (PI3K) (Scitable, 2014; Massie & Mills, 2006). Detailed information about other growth factor signaling pathways is available elsewhere (Popovic & Wilson, 2018; Lemmon & Schlessinger, 2010; Massie & Mills, 2006).

Figure 9. Schematic illustrating the central role of growth factor receptors binding to growth factors, and the subsequent activation of cell signaling pathways leading to changes in cell behavior.

As stated in the beginning of this section, diverse interactions occur among cells, growth factors and ECM in the in vivo cell microenvironment. Knowledge gained from these interactions could be applied for creation of bioactive biomaterials. An example for creation of such bioactive biomaterials is stated in the section 1.8.4 presented in this thesis.

1.8 Overview of tissue engineering The term "tissue engineering" was used for the first time in 1988 at a National Science Foundation workshop (Heineken & Skalak, 1991). This term highlights the creation of a new scientific discipline, which focuses on the

generation of tissues from cells with the help of biomaterials and growth factors (Akter, 2016; Langer & Vacanti, 1993; Heineken & Skalak, 1991). Even though the tissue engineering field is relatively new, the basic idea of tissue replacement seems to be given already by Gasparo Tagliacozzi (1546- 99), a Professor of Surgery and Anatomy at the University of Bologna (O'Brien, 2011). Tissue engineering is a phrase that can be used almost interchangeably with regenerative medicine (O'Brien, 2011). Historically, human use of materials to improve or restore the tissue dates to about 1065-740 BC, where a prosthetic wooden toe was used to structurally replace an amputated toe (Huebsch & Mooney, 2009). Damage to tissues or organs occasionally occur, for example due to trauma, ageing, cancer or congenital diseases (Akter, 2016; Huebsch & Mooney, 2009; Griffith & Naughton, 2002). Over the past several years, innovative medical devices has been developed with the strive to improve quality of patient’s life who are suffering from tissue or organ related diseases (Aramwit et al., 2017). Currently, there exists some treatment procedures, including surgical repair, drug therapy and transplantation (Akter, 2016). However, available treatment procedures have not necessarily decreased patient's mortality. Tissue loss caused due to traumatic or non-traumatic injury can be treated by autografting and allografting (Murugan & Ramakrishna, 2005). In autografting method, a tissue taken from one site of the donor is transplanted to another site of the same donor. Whereas in allografting method, a tissue taken from one individual is transplanted to other individual of the same species but of non-identical genetic composition (Murugan & Ramakrishna, 2005; Ambrosio et al., 2001). Albeit considered good, use of autografts is limited in the clinics due to the shortage of available donor sites. On the other hand, use of allogenic graft is prone to elicit immunogenic reactions and graft rejection, and may also transmit pathogens to the host (Murugan & Ramakrishna, 2005). To address aforementioned limitations, nearly two and a half decades ago the concept of tissue engineering was introduced with the aim to improve the quality of patient’s life (Langer & Vacanti, 1993). In the first part of this section, the rationale of tissue engineering is described. Thereafter, characteristics of a biomaterial and a brief description about applicability of different biomaterials for tissue engineering are described further. Another part of this section is devoted to describing the necessity and delivery of growth factors for tissue regeneration. Finally, challenges and

advances in the field of tissue engineering with a focus on skin tissue engineering are briefly presented.

1.8.1 Rationale of tissue engineering The rationale of tissue engineering (TE) is to combine cells isolated from the patient or donor with appropriate biomaterials (scaffolds) and suitable biochemical factors (growth factors) to improve or restore biological functions (Khademhosseini & Langer, 2016; Langer & Vacanti, 1993). According to the breakthrough paper in the field, tissue engineering is defined as “an interdisciplinary field of research that applies both the principles of engineering and the phenomena of the life sciences toward the development of biological substitutes that maintain, restore, or improve function of the tissue” (Langer & Vacanti, 1993). Clinical success of any tissue engineering approach is dependent on four key factors: 1) the cells that generate the tissue, 2) the biomaterial, which acts like a scaffold to provide structural support to cells, 3) the biochemical signaling molecules (growth factors) that regulate the cellular outcomes, 4) cell-biomaterial interactions, which help in tissue remodeling and development. Thus, the goal to generate functional tissues in vitro could possibly be achieved upon combining anchorage dependent cells with appropriate bioactive 3D scaffolds. In this manner the conditions provided to the cells are expected to recapitulate the in vivo microenvironment, thereby resulting in proper tissue formation under in vitro conditions (O'Brien, 2011; Langer & Vacanti, 1993). The standard paradigm of tissue engineering is illustrated in Figure 10. Briefly, cells derived from the patient or donor are expanded under in vitro conditions. Later, expanded cells are subsequently combined with soluble growth factors plus 3D scaffolds (or) with 3D scaffolds containing immobilized growth factors (bioactive 3D scaffold) to promote differentiation to a tissue. The engineered tissue construct is then implanted into the patient for tissue regeneration (Figure 10). In case of in situ regeneration, a 3D scaffold or a bioactive 3D scaffold is placed directly at the site of injury to possibly restore tissue formation by the recruitment of host cells (Griffith & Naughton, 2002).

Figure 10. Outline of tissue engineering. (i) Isolation of cells from the patient or donor. (ii) Expansion of isolated cells under in vitro conditions. (iii) Cell culture in either 3D scaffold or bioactive 3D scaffold. (iv) Differentiation of ex vivo engineered cell scaffold to a tissue. (v) Implantation of engineered tissue into the patient.

1.8.2 Characteristics of a biomaterial for its use in tissue engineering National Institutes of Health (NIH) defines biomaterials as, “any substance (other than a drug) or combination of substances synthetic or natural in origin, which can be used for any period of time, as a whole or part of a system which treats, augments, or replaces tissue, organ or function of the body” (Williams, 2009). The selection of biomaterials plays an important role in the field of tissue engineering (TE). Wanted characteristics of a biomaterial to be used as a scaffold for TE includes 1) certain degree of porosity, to allow cell infiltration, and diffusion of nutrients and waste products out of the scaffold; 2)

biocompatibility, meaning that the scaffold should have certain surface chemistry to promote adhesion, migration, proliferation, differentiation of the cells; 3) adequate mechanical properties in order to function from the time of implantation to the completion of tissue remodeling; 4) controlled biodegradability in order to allow endogenous host cells to create their own ECM; 5) the capability to recapitulate ECM to promote appropriate tissue formation under in vitro conditions (O'Brien, 2011; Hutmacher, 2000; Babensee et al., 1998).

1.8.3 Biomaterials in tissue engineering In the past 30 years, the use of biomaterials in tissue engineering is promptly progressing with the pursuit of creating novel medical devices to support or restore the function of natural tissues (Guarino et al., 2018; Bhat & Kumar, 2013). Based on the origin of biomaterials, they are generally classified as natural and synthetic materials, for their use as scaffolds in both hard and soft tissue engineering. Materials obtained from a natural source includes e.g. collagen, elastin, silk, Matrigel®, alginate and chitosan. Whereas materials obtained from a synthetic source includes e.g. ceramics and polymers. Detailed description about relevance of natural materials in tissue engineering applications is stated elsewhere (Gomes et al., 2013). Briefly, the use of a natural material as a biomaterial for tissue engineering applications is advantageous due to its biocompatibility and biodegradability (Lutolf & Hubbell, 2005). Nonetheless, batch-to-batch variations and risks of viral contamination that would cause immunogenicity are the challenges involved with the use of natural materials in tissue engineering (Ren et al., 2015; Nair & Laurencin, 2006). Additionally, the exact composition of certain natural materials, such as e.g. Matrigel® is not defined and thus will not allow complete control over the performed experiments, in addition to the large batch-to-batch variations. Ceramic scaffold e.g. tri-calcium phosphate, hydroxyapatite, is an example of a synthetic material and commonly used for hard tissue engineering. The low elasticity and high mechanical stiffness of ceramic scaffolds make them attractive as biomaterial for bone regeneration applications (Hench, 1998). Nonetheless, poor infiltration of bone cells inside the hydroxyapatite scaffold is a problem that needs to be addressed (O'Brien, 2011). This issue could possibly be solved to some extent by increasing the channel diameter within hydroxyapatite scaffolds (Rose et al., 2004). Another group of synthetic

materials are the synthetic polymers including poly-l-lactic acid (PLLA), polyglycolic acid (PGA), and poly-methyl methacrylate (PMMA), which were previously evaluated as scaffolds in tissue engineering (Peppas & Khademhosseini, 2016; O'Brien, 2011). For example, PMMA has been in use for hard tissue engineering applications, such as dental implants and hip replacements. However, this polymer may cause complications in the long run, probably due to left over monomers from the polymerization process, reaction catalysts or initiators. It was previously reported that the residual monomers of methyl methacrylate from PMMA possibly cause damage to eyes and skin (Gupta et al., 2012). It has also been reported that biodegradation of synthetic polymers like PLA and PGA can result in a decrease of local pH, thereby affecting cell growth (Agrawal & Athanasiou, 1997). Recent technological advancements in the field of genetic engineering have enabled creation biomaterials based on recombinant proteins such as elastin- like materials and silk-like materials. The possibility to customize biomaterials with bio-functional peptides has expanded the applicability of protein biomaterials for tissue engineering applications (Mulyasasmita & Heilshorn, 2011).

1.8.4 Role of growth factors in tissue engineering The clinical success of engineered tissue grafts is limited. One of the reasons is lack of sufficient vascularization that result in improper supply of nutrients and/or hypoxia deeper in the engineered tissue grafts (Novosel et al., 2011; Rouwkema et al., 2008). In vivo, supply of oxygen and nutrients to the tissues, as well as removal of waste products from the tissues, are performed by the vascular system via blood vessels, which further divide into capillaries. The maximum distance between two capillaries in vivo is 200 µm (Figure 11). It has been shown that cells remain viable by oxygen diffusion when they are located within a distance of 200 µm from the source of oxygen (Novosel et al., 2011; Rouwkema et al., 2008).

Figure 11. Schematic illustration of in vivo diffusion and transport processes to and from the tissue, and representation of maximum distance between two capillaries. Figure inspired from (Novosel et al., 2011).

Insufficient supply of oxygen and nutrients in engineered tissue constructs with thickness more than 200 µm is an important challenge to be addressed for advancement of tissue engineering (Rouwkema et al., 2008). The hypoxic condition created in the deeper region of engineered tissue constructs/grafts can stimulate vessel ingrowth by the release of angiogenic growth factors (Laschke et al., 2006). Nonetheless, vessel ingrowth induced by the released angiogenic growth factors is a slow process, and will not warrant the instant supply of nutrients required by the cells present in the deeper regions of engineered tissue grafts (Rouwkema et al., 2008). This suggests that in vitro creation of a pre- vascularized engineered tissue graft is an essential feature to be considered in tissue engineering (Baldwin et al., 2014; Lovett et al., 2009). One way to achieve pre-vascularization is by incorporation of angiogenic growth factors to recruit endothelial cells from the surrounding tissue or to stimulate endothelial cells that were added to the biomaterial. For example, to include VEGF and bFGF as an integral part of the biomaterial (Figure 10 (iii)). The main limitation when incorporating soluble growth factors is their low stability in the cell microenvironment. This means that a majority of the growth factor molecules will be degraded before reaching their target (Lee et al., 2011). To

compensate for their short half-life, frequent supplementation with large amounts of soluble growth factors is needed. Nonetheless, such large amounts growth factors can cause damage to the cells (Lee et al., 2011). Alternatively, retention and stability of growth factors can be achieved by immobilization on biomaterials (Briquez et al., 2015; Hajimiri et al., 2015). Growth factors can e.g. be covalently immobilized into a biomaterial using chemical conjugation methods. However, the use of chemicals can comprise the biological activity of the growth factor (Briquez et al., 2015). This could be overcome by using a chemical-free, gene fusion method, where the gene encoding a functional protein is fused to the gene for a self-assembling proteins. This has been demonstrated for e.g. Ultrabithorax, a recombinant transcription factor derived from Drosophila melanogaster (Huang et al., 2011) and for the partial spider silk protein 4RepCT, derived from the African nursery spider Euprosthenops australis (Stark et al., 2007). One possible approach to deliver a growth factor (bFGF was used as a model growth factor) that is covalently immobilized to 4RepCT is described in Paper III, in this thesis. Potential applications of angiogenic growth factors after their immobilization to self-assembling proteins are illustrated in the Figure 12. As described in section 1.7.3, certain growth factors, e.g. FGF-2, VEGF and TGF-β1, bind to the ECM via heparan sulfate proteoglycans (Schultz & Wysocki, 2009). Inspired from this information, researchers have modified biomaterials to contain growth factor binding sites, such as heparin or heparan sulfate-mimetic molecules, thereby enabling non-covalent, affinity based, immobilization of growth factors (Briquez et al., 2015). For example, in one study, collagen sponges impregnated with fibrin/heparin were used for the delivery of non-covalently immobilized FGF-2 (DeBlois et al., 1994). In a mouse model, subcutaneous implantation of collagen sponges pre-impregnated with a fibrin/heparin/FGF-2, resulted in tissue ingrowth within the implanted collagen sponges (DeBlois et al., 1994). A strategy for non-covalent immobilization of growth factors via spider silk as a biomaterial is described in Paper I. Briefly, biotinylated growth factor was captured on bioactive silk film, containing monomeric streptavidin genetically fused to 4RepCT (M4- 4RepCT).

Figure 12. Potential applications of angiogenic growth factors upon immobilization to partial spider silk scaffold.

2 Present investigations

2.1 Aims of the thesis The main aim of the present thesis is to immobilize different affinity and bioactive domains to partial spider silk proteins via genetic engineering. Moreover, to study the maintained functional properties of the silk part and added affinity/bioactive domains in silk fusion proteins. The specific aims of this thesis have been:

 Paper I: To investigate the possibility to functionalize 4RepCT silk protein with different fold-dependent affinity protein domains using genetic fusion/linkage followed by functional studies of silk materials generated from the silk fusion proteins.

 Paper II: To covalently combine two scFvs to 4RepCT and NTCT silk protein variants at the gene level and to evaluate the possibility to use scFv-4RepCT/NTCT silk fusion proteins as capture probes for generation of sensitive immunoassays.

 Paper III: To bioactivate 4RepCT silk protein with a pleotropic growth factor, bFGF and evaluate the possibility to use silk-bFGF scaffold for in vitro cell culture and tissue engineering applications.

 Paper IV: To immobilize two affibody-based binders of VEGFR2 by genetic fusion to 4RepCT and to evaluate the maintained bioactive properties of immobilized affibodies by performing cell culture studies.

2.2 Results and discussion

2.2.1 Paper I: Recombinant spider silk genetically functionalized with affinity domains. In paper I, functionalization of 4RepCT with various affinity domains was evaluated. Four different fold dependent affinity domains (Z, C2, ABD and M4) were covalently attached to the N-terminus of 4RepCT at the gene level (Figure 13a). Domains Z (7 kDa) (Nilsson et al., 1987) and ABD (5 kDa) (Kraulis et al., 1996) are folded into three helix bundles, and bind to IgG and albumin respectively. In contrast, C2 (6 kDa) and M4 (17 kDa) domains contain β/α structures (Sauer-Eriksson et al., 1995) and a β-sheet barrel (Wu & Wong, 2005) respectively, and have affinity towards IgG and biotin, respectively. All four recombinant silk constructs were successfully produced in the BL21 (DE3) strain of E. coli and purified using immobilized metal ion affinity chromatography (IMAC) under non-denaturing conditions (Figure 13b). It is worth noting that the produced silk fusion proteins could still be self-assembled to film and fiber format despite the relatively large sizes (5-17 kDa) of the affinity domains added to the 4RepCT (Figure 13c). Moreover, no additional processing steps are required to make fibers and films of silk fusion proteins. Since target binding ability of the added affinity domains is dependent on their secondary structure, Fourier transform infrared spectroscopy (FTIR) analysis was performed on films and fibers made from all silk fusion proteins. The results from FTIR analysis supported a maintained β-sheet structure, which is a characteristic of 4RepCT, in both film and fiber formats of silk fusion proteins. In order to evaluate the selective IgG binding ability of Z and C2 domains, a complex serum mixture was added to the fibers and films of Z-4RepCT and C2-4RepCT. After wash, bound IgG was released and analyzed using SDS- PAGE, which showed a band corresponding to the correct size of IgG (140 kDa), thus confirming retained functionality of added Z and C2 domains to selectively bind IgG. Likewise, the functionality of added ABD was verified upon incubation of complex human plasma to fiber and film of ABD-4RepCT. Release of bound albumin and gel analysis confirmed the maintained functionality of ABD to selectively bind albumin.

Figure 13. Production of soluble functionalized silk-fusion proteins and characterization of the silk-like materials formed thereof. (a) Schematic representations of four different functionalized silk fusion proteins, Z-4RepCT (32 kDa), C2-4RepCT (31 kDa), ABD-4RepCT (30 kDa), and M4- 4RepCT (41 kDa). Secondary structure elements of the functionalization domains (Protein Data Bank ID: 2spz, 1fcc, 1gjt, and 1swe, respectively) are color-coded; α-helix in light blue, β-sheet in purple. (b) SDS-PAGE of eluted fractions from immobilized metal ion affinity chromatography (IMAC) of four soluble functionalized silk fusion proteins, and 4RepCT. Target proteins are indicated by black squares. (c) Light microscopy images of films and fibers of functionalized silk fusion protein, compared to those of 4RepCT. Scale bars: 1mm. Adapted with permission from (Jansson et al., 2014). Copyright © 2014 American Chemical Society.

In order to explore the possibility to create multi-functional silk materials, mixed films and mixed fibers were prepared by combining soluble fractions of

Z-4RepCT and ABD-4RepCT (Figure 14a). The ability of mixed films and fibers to selectively capture both IgG (by Z-4RepCT) and albumin (by ABD- 4RepCT) was shown by addition of human plasma to the mixed silk materials followed by the release of both bound target molecules, as shown by gel electrophoresis, thus confirming the concept of multi-functional silk materials (Figure 14b).

Figure 14. Multi-functionalized silk materials can simultaneously bind two targets. (a) Light microscopy images of films and macroscopic fibers prepared by mixing soluble Z-4RepCT with soluble ABD-4RepCT protein. Scale bars: 1 mm. (b) The ability of mixed Z- 4RepCT/ABD-4RepCT film and fiber to simultaneously bind IgG (via the Z domain) and albumin (via the ABD domain). Human blood plasma served as source for both IgG and albumin. Released fractions from mixed film and mixed fiber were analyzed by SDS-PAGE. Bands of IgG and albumin are indicated with arrows. Adapted with permission from (Jansson et al., 2014). Copyright © 2014 American Chemical Society.

Due to the simplicity and versatility of the biotin-streptavidin system, it has gained a significant focus in the field of biotechnology (Diamandis & Christopoulos, 1991). It has also been extensively used to link functional groups to biomaterials (Wang & Kaplan, 2011; Spieß et al., 2010). Likewise, the possibility to covalently link the monomeric streptavidin domain (M4) to 4RepCT in the quest to link functional groups to silk based materials has been verified in Paper I. To demonstrate the biotin binding ability of the M4 domain in the M4-4RepCT construct, films of M4-4RepCT were incubated with biotinylated DNA molecules. High affinity between monomeric streptavidin

and biotinylated ligands (Wu & Wong, 2005) resulted in a difficulty to release the bound biotinylated ligands. Since release of bound biotinylated ligands usually requires harsh elution conditions (Rybak et al., 2004), a restriction enzyme site was included at the 5’ end of the biotinylated DNA molecules to ensure mild release, which could be confirmed by analysis using a DNA gel. Thus the applicability of M4-4RepCT silk materials for nucleic acid hybridization assays is envisioned. Furthermore, the use of M4-4RepCT films to immobilize growth factors was evaluated (Figure 15a). This concept was confirmed by capture of EGF molecules pre-labeled with a chromophoric biotin onto M4-4RepCT films, followed by measurement of the absorbance signal from the bound biotinylated EGF molecules (Figure 15b).

Figure 15. Silk films functionalized with monomeric streptavidin (M4-4RepCT) for presentation of growth factors. (a) Schematics for presentation of biotinylated EGF onto M4-4RepCT. Bound EGF is detected by measuring absorbance of chromophoric biotin (b) Graph showing signal from chromophoric biotin-EGF to M4-4RepCT and 4RepCT control films, respectively. Adapted with permission from (Jansson et al., 2014). Copyright © 2014 American Chemical Society.

In a recent study, a recombinant silk construct named FN-4RepCT was created, where a RGD cell binding peptide derived from fibronectin was genetically fused to 4RepCT and was presented in a loop format, thereby enhancing the cell supportive ability of spider silk (Widhe et al., 2016). Upon combining soluble FN-4RepCT and M4-4RepCT, an advanced silk based cell scaffold could possibly be designed for in vitro cell culture studies, provided that the basal culture medium is supplemented with biotinylated growth factors. Moreover, due to the high affinity between M4 domain and biotinylated

ligands (Wu & Wong, 2005), long term use of bound biotinylated growth factors is envisioned during media change, thus providing an economic benefit. On the other hand, if release of growth factors is demanded by the cells and if it is difficult to biotinylate growth factors, a reversible two-step presentation of growth factor via Z-4RepCT materials could instead be employed. The feasibility of such two-step approach using Z-4RepCT silk was shown by the capture of the vascular endothelial growth factor (VEGF) using anti-VEGF IgG, which was first bound to the Z domain. To visualize the indirect binding of VEGF to Z-4RepCT films via anti-VEGF IgG, the VEGF was biotinylated and a streptavidin labelled with fluorophore was used for detection. Since it is challenging to determine the specific function of biotinylated growth factors captured to 4RepCT fusions, a domain with more easily measured functionality, the enzyme xylanase, was biotinylated and captured onto M4-4RepCT films. Activity of bound xylanase was thereafter verified upon conversion of the substrate to a product that could be measured at a certain wavelength (Figure 16a). Higher absorbance signal was observed from M4-4RepCT films with biotinylated xylanase than corresponding Z-4RepCT control films, thus confirming the maintained activity of the xylanase (Figure 16b). This finding highlights the possible use of M4-4RepCT silk materials to capture also other enzymes, which have potential applications in the field of biology and medicine (Costa et al., 2005).

Figure 16. Silk films functionalized with monomeric streptavidin (M4-4RepCT) for presentation of an enzyme. (a) Schematics showing presentation of a biotinylated enzyme on M4-4RepCT silk, where the activity of bound xylanase is determined by measuring its ability to convert substrate to a colored product. (b) Graph showing absorbance of the product obtained by biotinylated xylanase bound to M4-4RepCT and Z-4RepCT control films, respectively. Adapted with permission from (Jansson et al., 2014). Copyright © 2014 American Chemical Society.

2.2.2 Paper II: Genetic fusion of single-chain variable fragments to partial spider silk improves target detection in micro- and nanoarrays. In paper II, one of the attractive features of spider dragline silk proteins, namely stickiness (Vollrath, 2000) has been exploited for the development of high performing recombinant antibody based immunoassays. In this study, two single chain variable fragments (scFvs), αVEGF and αC1q (30 kDa) that bind to the serum proteins VEGF (low abundant) and C1q (high abundant) respectively, were selected from a human recombinant scFv phage-display library (Soderlind et al., 2000). Corresponding genes encoding the scFvs were then genetically attached to either ends of two partial spider silk variants, 4RepCT (herein denoted as RC) and NTCT (herein denoted as NC) (Figure 17a). All of the generated RC/NC silk fusion constructs (RC-α and α-RC, 52 kDa, NC-α and α-NC, 54 kDa) could be produced in E. coli and purified using immobilized metal ion affinity chromatography (IMAC) (Figure 17b,c). Even though a partial degradation of the target proteins was observed, scFv-silk fusion proteins of sufficient quality were obtained. Concentrated fractions of soluble scFv-RC/NC silk fusion proteins could be processed to silk-like fibers (Figure 17d). This confirmed a retained functionality of RC and NC silk to form silk-like fibers despite covalently attached to a scFv domain (30 kDa), which is larger in size compared to the sizes of both RC (23 kDa) and NC (27 kDa) silk proteins.

Figure 17. Description of scFv-silk fusion proteins. (a) Schematic representations of the different scFv-silk fusion proteins, RC-α (52 kDa), α-RC (52 kDa), NC-α (54 kDa) and α-NC (54 kDa). ScFvs are shown as α (30 kDa). (b, c) SDS-PAGE analysis, showing eluted protein fractions of RC-α, α-RC, NC-α and α-NC fusion proteins, respectively. Target protein bands are highlighted by rectangles. Additional bands submitted for mass spectrometry analysis are indicated by arrows or star. Repositioning of protein lanes are represented by vertical black lines. (d) Light microscopy images of fibers made from scFv- silk fusion proteins. Scale bar: 1 mm.

To investigate the target binding affinities of the added scFv domains in the scFv-RC silk constructs, a microarray slide carrying microspots of scFv-RC silk fusion proteins (RC-αVEGF & αVEGF-RC, RC-αC1q & αC1q-RC) was designed. Since silk protein provides a hydrophilic environment and mechanical stability to the array spot, this might change the morphology of the array spot and thereby affect the outcome of immunoassay. Therefore, mixed spot (soluble scFvs and RC added together) and double spot (soluble scFv added onto dried RC) formats were formulated to constitute controls of scFvs non-covalently attached to RC silk. Incubation of VEGF antigens onto a microarray slide revealed VEGF binding to the microspots containing αVEGF, RC-αVEGF and αVEGF-RC proteins but almost no binding to the other microspots (Figure 18a). Similarly, addition of C1q antigens to the microarray slide showed binding of C1q antigens only to the microspots of αC1q, RC- αC1q and

αC1q-RC (Figure 18b). These observations also confirmed that there exists no cross reactivity among different scFv-RC silk fusion proteins. Moreover, the target detection signal was distinctly increased when scFvs were covalently attached to RC silk compared to the same added amount of scFvs to the microspots containing only scFv. The increase in target binding signal might be due to the high propensity of spider silk proteins to self-assemble and adhere to the solid surfaces, thereby resulting in well-oriented antibody fragments from the fusion protein. Presence of well-oriented immobilized antibody fragments would enable efficient binding to the target molecules, thereby contributing to the success of solid-phase assays. Likewise, in another study, use of well- oriented antibody fragments has been shown to improve the target binding (Hu et al., 2013). Another reason for improved target detection signal might be that a higher proportion of scFvs might have stayed within the array spots during the several processing steps involved in the immunoassay when attached to the silk protein. However, very low target detection signal, close to the background signal, was obtained from mixed and double spots, thus demonstrating the necessity of having scFvs covalently linked to silk when used as capture probes in immunoassays.

Figure 18. Analysis of target antigen binding to scFv-RC silk fusion proteins in microarrays. (a) Graph plot demonstrating VEGF antigen binding to different formats of αVEGF and controls. (b) Graph plot displaying C1q antigen binding to various αC1q formats and controls.

Incubation of a non-fractionated serum sample onto the microarray slide containing microspots of scFv-RC silk fusion proteins confirmed the selective binding between the scFv-silk fusion proteins and their respective targets

present in the serum (Figure 19). This highlights the ability of scFv-RC silk fusion proteins to bind their targets even from a complex sample.

Figure 19. Detection of VEGF and C1q antigen from serum samples by scFv-RC silk fusion proteins in microarrays. Graph plot displaying VEGF and C1q antigen binding to α, RC-α, α-RC. RC and BSA are used as controls.

In the quest for generation of sensitive and high density arrays, possible use of scFv-silk fusion proteins as capture probes was investigated also using nanoarrays. Deposition of proteins for nanoarrays can be done by several techniques, for example using dip-pen technology (Petersson et al., 2014; Wingren & Borrebaeck, 2007). The repetitive region in the RC silk variant has a high tendency to fibrillate, which could potentially clog the nozzle if deposited using dip-pen technology. To avoid this problem, NTCT (NC), a new engineered variant of spider silk was generated, without including the repetitive region, and used for nanoarray analysis. Again the previously selected scFvs (αVEGF and αC1q) were covalently attached onto either ends, resulting in the generation of scFv-NC silk fusion proteins (NC-αVEGF & αVEGF-NC, NC- αC1q & αC1q-NC) (Figure 17a). The applicability of generated scFv-NC silk fusion proteins for creation of high density arrays, nanoarrays, was thus studied. Any variations in morphology of nanospots can affect the creation of high density arrays and array performance as well. However, no variations in spot morphologies was observed among nanospots made of scFv-NC silk fusion proteins, scFvs alone and control sample, as confirmed by microscopy analysis of the nanospots (Figure 20a). Similar to microarray analysis, upon addition of pure antigen samples (VEGF and C1q) onto the nanoarray slides containing nanospots of scFv-NC silk fusion proteins, no sign of cross reactivity was observed among different scFv-NC silk fusion proteins (Figure 20b, c).

Furthermore, significantly higher target detection signal was observed by scFvs when covalently attached to NC silk compared to the same added amount of scFvs alone (Figure 20b, c). Thus, the results from the nanoarray analysis were in line with the microarray results.

Figure 20. Investigation of target antigen binding to scFv-NC silk fusion proteins in nanoarrays. (a) Image showing morphology of nanospots of protein samples (BSA, scFv (α), NC-α and α-NC) applied using Dip Pen Nanolithography (DPN). (b) Graph plot showing VEGF antigen binding to αVEGF, NC-αVEGF, αVEGF-NC and controls (n.s., not significant; **p < 0.01). (c) Graph showing C1q antigen binding to αC1q, NC-αC1q, αC1q- NC and controls (*p = 0.05).

In order to rule out unspecific binding of serum components to the silk, C1q depleted serum was prepared. Upon addition of C1q depleted serum onto a scFv-silk coated nanoarray slide, very low target binding signal was reported from NC-αC1q & αC1q-NC, confirming low unspecific binding of serum components to scFv-NC silk proteins (Figure 21a). To study the array setup using a clinically relevant sample, pooled serum from Systemic Lupus Erythematosus (SLE) patients was examined. Upon incubation of serum from SLE patients onto the nanoarray slide, a higher target signal was reported by αVEGF-NC silk fusion protein compared to incubation with C1q depleted serum (Figure 21b). This might be due to the high levels of VEGF in serum derived from SLE patients (Zhou et al., 2014), which is an hallmark of patients with SLE disease, compared to low levels of VEGF present in the serum obtained from normal patients. The results from the nanoarray analysis confirmed a retained functionality of the two scFvs that were attached to NC silk.

Figure 21. Detection of target antigens from non-fractionated clinical samples by scFv-NC silk fusion proteins in nanoarrays. (a) Graph showing antigen binding to α, NC-α, α-NC when incubated with two concentrations of C1q depleted serum onto subarrays. NC silk without scFv was used as a control. (b) Graph displaying target antigen binding to α, NC-α, α-NC upon incubation of subarrays with two concentrations of serum collected from SLE patient (n.s., not signifcant; **p ≤ 0.01).

To summarize, in Paper II a procedure to immobilize antibody fragments via partial spider silk, which result in well-oriented antibody fragments, was demonstrated. Moreover, the use of scFvs covalently attached to the N- terminus of partial spider silk could yield stable scFvs within array spots. Thus, a future use of such in vitro targeting agents for sensitive diagnostics is envisioned.

2.2.3 Paper III: Bioactivation of Spider Silk with Basic Fibroblast Growth Factor for in vitro Cell Culture: A Step toward Creation of Artificial ECM. Previously, functionalization of the partial spider silk protein (4RepCT) with short cell binding peptides (~1.5 kDa) was investigated (Widhe et al., 2016; Johansson et al., 2015). In paper III, for the first time, the possibility to bioactivate 4RepCT with a bioactive domain, basic fibroblast growth factor (~17.2 kDa) was evaluated. Basic fibroblast growth factor (bFGF) is a pleotropic growth factor, which can regulate behavior of multiple cell types, including fibroblasts and endothelial cells (McGee et al., 1988; Schweigerer et al., 1987). Furthermore, bFGF possess affinity towards heparin and exhibit fold dependent activity (Wang et al., 1996). In this study, the gene encoding bFGF was genetically fused to the C-terminus of 4RepCT, thereby resulting in a silk- bFGF construct (Figure 22a). The corresponding silk-bFGF fusion protein was

produced in the BL21 (DE3) strain of E.coli and purified using IMAC under non-denaturing conditions (Figure 22b). The produced silk-bFGF fusion protein could self-assemble into a fiber, demonstrating that the beta barrel fold of bFGF did not interfere with the fiber forming ability of 4RepCT (Figure 22c).

Figure 22. Construction and purification of a silk-bFGF fusion protein. (a) Schematic illustration of the silk-bFGF construct. A glycine-serine linker (GS)3 is included between the silk part and bFGF. (b) SDS-PAGE analysis of the purified silk-bFGF fusion protein. Protein ladder is presented to the left. Target protein band (42 kDa) is indicated with an arrow. (c) Light microscopy image of a fiber generated from self-assembly of the silk-bFGF fusion protein. Scale bar: 1 mm. Adapted with permission from (Thatikonda et al., 2018). Copyright © 2018 American Chemical Society.

In this study, two different formats of silk-coatings, namely single-coat and double-coat were investigated. In single-coat, direct addition of the silk-bFGF fusion protein onto a surface was performed. While in double-coat, silk-bGF protein was added on top of surfaces previously coated with 4RepCT (WT- silk). To investigate the assembly of silk-bFGF fusion protein in real-time and the viscoelastic properties of thereof formed surface coatings, QCM-D measurements were performed (Figure 23). In single-coat format, a decrease in the oscillation frequency was observed upon flow of silk-bFGF fusion protein on a sensor surface modified to become hydrophobic. Thus, surface assembly of silk-bFGF protein into surface coatings was confirmed. Moreover, the adsorbed protein was not washed away during 10 min wash with Tris buffer (Figure 23a, (i)). In double-coat format, faster assembly of silk-bGF was observed, compared to the single-coat format (Figure 23a (ii)). This could be due to the favorable interactions occurring between silk-bFGF and WT-silk,

compared to less favorable interactions between silk-bFGF and the sensor surface (in single-coat). Viscoelastic properties of the surface coatings can be obtained from QCM- D measurements by calculation of dissipation (D) to frequency (f) ratio. This information can be used to determine the stiffness of the coatings, which previously has been shown to affect cell behavior (Wells, 2008). Silk-bFGF coatings obtained using the single and double-coat method generated ΔD/Δf values of 0.035 (Figure 23b, (i)) and 0.076 (Figure 23b, (ii)), respectively. Double-coat of silk-bFGF seems to be slightly more viscous compared to previously reported fibronectin (ΔD/Δf values of ~0.03) (Molino et al., 2012) and bovine serum albumin (< 0.01) (Molino et al., 2012).

Figure 23. Real time monitoring of silk-bFGF assembly into surface coatings. (a) Graphs from QCM-D measurements showing real time formation of single-coat (i) and double-coat (ii) of silk-bFGF. (b) Graphs obtained from QCM-D measurements showing frequency shifts in relation to dissipation shifts of QCM-D measurements (3rd overtone) of silk-bFGF as (i) single-coat and (ii) double-coat. The filled triangle represent the adsorption start of silk- bFGF while the open triangle represents the adsorption start of WT-silk. Dotted triangle represents the flow of Tris wash buffer. Adapted with permission from (Thatikonda et al., 2018). Copyright © 2018 American Chemical Society.

Complementary to QCM-D measurements, real time self-assembly of silk- bFGF fusion protein into surface coatings was further confirmed by surface plasmon resonance studies (SPR) (Figure 24).

Figure 24. Surface adsorption of silk-bFGF. SPR study demonstrating surface adsorption of single-coat silk-bFGF (a), and double-coat silk-bFGF, silk-bFGF on WT-silk (b). Filled triangle represents injection of silk-bFGF. Open triangle represents injection of WT-silk. Dashed triangle represents flow of 20mM Tris buffer containing 0.5 M NaCl. Adapted with permission from (Thatikonda et al., 2018). Copyright © 2018 American Chemical Society.

Staining with a fluorophore labeled antibody, which is specific toward the N-terminal histidine tag present in silk-bFGF confirmed that the double-coat format resulted in more uniform and homogeneous coating than single-coat of silk-bFGF (Figure 25a, b). Therefore, the double-coat format of silk-bFGF was used for receptor binding and cell culture studies. When examined by Atomic force microscopy (AFM), the nanotopography of silk-bFGF coatings revealed a fibrillar network (Figure 25c). Thus, the prepared silk coatings mimics the fibrillar networks present in the extracellular matrix rather than a flat coating. Additionally, the silk part will anchor the silk- bFGF fusion protein in an orientation away from the hydrophobic surface, thus protecting the growth factor from denaturation when exposed to a hydrophobic surface.

Figure 25. Fluorescence staining and AFM imaging of silk-bFGF coatings obtained by self- assembly onto a solid surface. (a) Representative fluorescence micrographs from staining of different silk coatings with a Alexa488 labeled antiHis6 antibody. Scale bar: 100 μm. (b) Graph plot of fluorescence intensities from staining of silk-bFGF coatings with an Alexa488 labeled antiHis6 antibody. The anti-His6 antibody was used to discriminate between silk- bFGF (containing the His6-tag) and WT-silk (which does not contain the His6-tag). Single- coat silk-bFGF vs single-coat WT-silk (*p < 0.05), single-coat silk-bFGF vs double-coat silk-bFGF (**p < 0.01), double-coat silk-bFGF vs single-coat WT-silk (***p < 0.001). (c) AFM images depicting the nanotopography of single-coat and double-coat formats of silk- bFGF, and single-coat of FN-silk and WT-silk. Adapted with permission from (Thatikonda et al., 2018). Copyright © 2018 American Chemical Society.

A step toward creation of an artificial ECM was tested by combining silk- bFGF with FN-silk, an engineered spider silk protein created by genetic fusion of a RGD cell binding peptide derived from fibronectin to 4RepCT which was previously reported to support cell cell adhesion (Widhe et al., 2016). In such combined silk coatings, cells are anticipated to adhere onto FN-silk and at the same time proliferate in response to the bFGF presented in silk-bFGF. In order to verify the applicability of combined silk coatings for in vitro cell culture studies, adhesion of primary endothelial cells (HUVECs) on different silk coatings was studied in MII culture medium, containing 0.5% FBS and no added growth factors. Crystal violet staining of adhered cells revealed significantly higher cell adhesion on combined silk coatings compared to other silk coatings (Figure 26a).

Figure 26. Adhesion to and proliferation of primary endothelial cells on different silk coatings. (a) Representative graph plot of the quantified area of cells adhered to different silk coatings. Silk-bFGF, FN-silk, silk-bFGF+FN-silk vs WT-silk (****p < 0.0001), silk- bFGF, FN-silk vs silk-bFGF+FN-silk (****p < 0.0001). (b) Representative graph plot from Alamar blue viability assay demonstrating proliferation of HUVECs on coatings of silk- bFGF (double-coat), FN-silk (single-coat), silk-FGF+FN-silk (double- coat), WT-silk (single-coat), or non-coated culture well (tissue culture treated) in MII medium (contains 0.5% FBS and no supplemented growth factors). Silk-bFGF+FN-silk vs silk-bFGF, FN-silk, TCT, WT-silk (on Day 2,*p < 0.05; on Day 4, ***p < 0.001; on Day 6, ****p < 0.0001). Adapted with permission from (Thatikonda et al., 2018). Copyright © 2018 American Chemical Society.

In order to explore the applicability of combined silk coatings for 2D cell culture studies, cell viability on different silk coatings was studied using Alamar blue and live/dead viability assays. Alamar blue assay demonstrated that a double-coat format of combined silk-bFGF+FN-silk supported significantly higher proliferation of endothelial cells when compared to other silk coatings and the uncoated TCT control (Figure 26b). The promising results from 2D cell culture studies motivated us to test the relevance of combined silk materials as a tissue engineering scaffold for 3D cell culture. Cells, growth factors and scaffolds are generally considered as the tissue engineering triad (Langer & Vacanti, 1993). As previously discussed in section 1.8.4, cells in deeper region of thicker engineered tissue graft (>200 µm) can experience hypoxia, mainly due to lack of sufficient vascularization. One way to limit hypoxia is by promoting vascularization within engineered tissue grafts. This can be possible by bioactivation of engineered tissue grafts with angiogenic growth factors, e.g. bFGF (O'Brien, 2011). The feasibility to use a multi-functional silk fiber based scaffold bioactivated with growth factors (by

silk-bFGF) and cell binding peptides (by FN-silk) was verified in this study. Primary endothelial cells that were cultured integrated within a multi- functional silk fiber scaffold demonstrated significantly higher proliferation compared to cells in a FN-silk fiber (Figure 27a), and better spreading (Figure 27b) when cultured in medium with low serum and no growth factors. These results encourage the future prospect to create a vascularized tissue engineering scaffold based on functionalized silk.

Figure 27. Culture of endothelial cells (HUVECs) integrated in fibers of silk-FGF+FN-silk (combined) and FN-silk (control). (a) Representative graph plot from Alamar blue viability assay demonstrating proliferation of HUVECs after integration into silk-bFGF +FN-silk (combined) and FN-silk (control) fibers in medium with low serum and no supplemented growth factors. Silk-bFGF+FN-silk vs FN-silk (****p < 0.0001). (b) F-actin (green) and nuclei (blue) staining of HUVECs integrated within combined and control fibers after 5 days of culture in medium with low serum and no supplemented growth factors. Scale bar: 100 μm. Adapted with permission from (Thatikonda et al., 2018). Copyright © 2018 American Chemical Society.

To summarize, in Paper III a strategy to bioactivate partial spider silk with growth factors was reported. As a model growth factor, bFGF was genetically fused to the partial spider silk protein. Obtained silk-bFGF fusion proteins could self-assemble into surface coatings and to fibers. Moreover, possibility to create artificial ECM was verified by combining silk-bFGF and FN-silk proteins and using them as scaffolds for 2D and 3D cell culture (Figure 28). Use of such multi-functional silk-based scaffolds for creation of vascularized engineered tissue constructs is thus envisioned for regenerative medicine applications.

Figure 28. Schematic representation of multi-functional silk based scaffolds, silk-bFGF and FN-silk, for 2D and 3D cell culture studies.

2.2.4 Paper IV: Artificial VEGFR2-Specific Growth Factors Demonstrate Agonistic Effects in Both Soluble Form and When Immobilized Via Spider Silk. In Paper IV, the possibility to use two affibody-based binders as artificial ligands for VEGFR2 signaling was first explored. The herein generated affibody-based binders could recognize different paratopes on VEGFR2. The individual VEGFR2 binding entities were combined two times in a dimeric construct, Zdimer (Figure 29a), and four times in a tetrameric construct, Ztetramer (Figure 29b).

Figure 29. Schematics of affibody-based binders for VEGFR2. The affibody entities were combined two times as Zdimer (a) and four times as Ztetramer (b). Triple helix structure retrieved from Protein Data Bank ID: 2spz.

Previously, in vitro cell culture studies showed that soluble Zdimer resulted in inhibition of VEGFR2 phosphorylation and proliferation of HEK293/KDR cells (Fleetwood et al., 2016), thus, confirming the antagonistic nature of soluble Zdimer towards VEGFR2 signaling. The receptor phosphorylation studies herein performed using soluble Ztetramer resulted in activation of

VEGFR2 phosphorylation, thereby, confirming the agonistic nature of soluble

Ztetramer towards VEGFR2 signaling. Observed cellular outcomes of soluble Zdimer and Ztetramer were in line with the illustrations presented in Figure 30a. The affibody constructs, Zdimer and Ztetramer, were then fused genetically to the partial spider silk protein, 4RepCT with the aim of generating agonistic artificial ligands for VEGFR2 signaling. As reported earlier, partial spider silk (4RepCT) dimerizes in solution (Nilebäck et al., 2017). We hypothesized that silk dimerization might result in an orientation of two dimeric affibodies, thus mimicking tetrameric affibodies, which thereby could result in agonistic properties (Figure 30b, (i)). Possible cell interactions of Zdimer and Ztetramer after their immobilization via genetic fusion to silk are illustrated in Figure 30b.

Figure 30. Schematics of possible cell interactions of soluble and immobilized affibody constructs. (a) Antagonistic effect of soluble Zdimer (i) and agonistic effect of soluble Ztetramer. (b) Antagonistic and/or agonistic effect of immobilized Zdimer (i) and agonistic effect of immobilized Ztetramer via fusion to silk.

Briefly, DNA sequences encoding Zdimer and Ztetramer were genetically fused to the gene encoding the 4RepCT protein thereby resulting in Zdimer-silk and Ztetramer-silk recombinant constructs (Figure 31a). SDS-PAGE analysis confirmed the integrity of the purified Zdimer-silk and Ztetramer-silk fusion proteins (Figure 31b). Additionally, the ability of silk fusion proteins to form

fibers (Figure 31c) was verified by combining purified Zdimer-silk or Ztetramer- silk with FN-silk, an engineered spider silk protein that was previously shown to support cell adhesion (Widhe et al., 2016).

Figure 31. Construction and purification of VEGFR2 specific affibodies immobilized via partial spider silk. (a) Schematics of Zdimer-silk and Ztetramer-silk constructs. (b) SDS-PAGE analysis of the purified Zdimer-silk and Ztetramer-silk fusion proteins. Target protein bands are pointed with arrows. (c) Light microscopy images of fibers made by combining Zdimer-silk or Ztetramer-silk, and FN-silk. Scale bar: 1 mm.

The ability of Zdimer-silk and Ztetramer-silk fusion proteins to assemble into surface coatings was confirmed by QCM-D measurements (Figure 32a). In a recent study, we showed that addition of a silk fusion protein as a top coating on surfaces earlier coated with WT-silk protein gave rise to more homogenous coatings, compared to direct coating of the silk fusion protein (Thatikonda et al., 2018). For this reason, Zdimer-silk and Ztetramer-silk fusion proteins added on top of surfaces previously coated with WT-silk were used in this study. QCM- D results confirmed the assembly of both silk fusion proteins into surface coatings (Figure 32a). However, the interactions between WT-silk and Ztetramer- silk (Figure 32a, (ii)) appeared to be less efficient compared to WT-silk and

Zdimer-silk interactions (Figure 32a, (i)). This might be due to possible interference on silk assembly caused by the large size of Ztetramer compared to Zdimer.

VEGFR2 binding to formed surface coatings of silk fusion proteins was studied by surface plasmon resonance (SPR) analysis. The results from SPR confirmed a higher receptor binding onto Zdimer-silk and Ztetramer-silk coatings compared to WT-silk coating (control) (Figure 32b). However, a lower receptor binding was observed for Ztetramer-silk (Figure 32b, (ii)) compared to the Zdimer-silk coating (Figure 32b, (i)). This might be due to a lower adsorption of Ztetramer-silk on WT-silk coating (Figure 32, (ii)) which resulted in lower receptor binding. Thus, results from SPR were correlated with the QCM-D measurements.

Figure 32. Self-assembly of Zdimer-silk and Ztetramer-silk fusion proteins into surface coatings and receptor binding to thereof formed coatings. (a) Graphs from QCM-D measurements showing formation of coatings made of Zdimer-silk (i) and Ztetramer-silk (ii) fusion proteins on surfaces previously coated with WT-silk. The third frequency overtones is shown. Open triangle indicates injection of WT-silk, whereas closed triangle indicates injection of Zdimer- silk (in (i)) or Ztetramer-silk (in (ii)) fusion proteins. Dotted triangle indicates injection of 20 mM Tris wash buffer. (b) VEGFR2 receptor binding to coatings made of Zdimer-silk (solid line, (i)) and Ztetramer-silk (solid line, (ii)). Dotted line represents VEGFR2 receptor binding to WT-silk coating (i and ii). Arrow represents injection of VEGFR2.

To verify the relevance of Zdimer-silk and Ztetramer-silk coatings for in vitro cell culture studies, receptor phosphorylation and proliferation of

HEK293/KDR cells were studied. In order to improve cell adhesion to coatings of Zdimer-silk and Ztetramer-silk proteins, they were combined with the FN-silk protein, an engineered spider silk protein previously reported to support cell adhesion (Widhe et al., 2016). Results from the phosphorylation assay demonstrated that cells cultured on Ztetramer-silk coatings showed highest level of phosphorylated VEGFR2 receptor. Moreover, Zdimer-silk coatings demonstrated higher levels of phosphorylated receptor compared to FN-silk and WT-silk coatings. Cell proliferation on different silk coatings was studied using the Alamar Blue viability assay. Results from the assay performed on day

2, 4, 6 and 8 showed higher cell activity on Zdimer-silk and Ztetramer-silk coatings compared to non-functional silk, WT-silk coating. As hypothesized earlier, results from phosphorylation and proliferation assay thus confirmed the agonistic effect of Zdimer and Ztetramer for VEGFR2 signaling, when genetically fused to the partial spider silk protein. One challenge in tissue engineering is to provide supply of nutrients and removal of waste products from the cells located in the deeper regions of a engineered tissue constructs (>200 µm thickness) (Novosel et al., 2011). This problem could be circumvented by creation of a vessel-like network within the engineered tissue construct. In this study, a possibility to create vessel-like structures was verified by co-culture of endothelial cells and fibroblasts in FN- silk foams containing Ztetramer-silk and FN-silk fibrils. To summarize, in Paper IV, site-specific and covalent immobilization of dimeric and tetrameric affibodies, which possesses affinity for VEGFR2, was achieved by gene fusion to a partial spider silk protein. Purified Zdimer-silk and Ztetramer-silk fusion proteins could self-assemble into coatings and fibers. Moreover, retained VEGFR2 receptor binding onto Zdimer-silk and Ztetramer-silk coatings was confirmed. Results from in vitro cell culture studies confirmed the retained VEGFR2 cell signaling agnositic effect exhibited by dimeric and tetrameric affibodies after immobilization via silk. Moreover, the possibility to achieve a vessel-like network in a silk based cell scaffold was verified upon use of Ztetramer-silk fibrils. The use of such materials to promote vasculature within cell scaffolds could potentially be used in tissue engineering applications.

3 Concluding remarks and future perspectives

The studies presented in this thesis were focused on functionalization of a partial spider silk protein, 4RepCT, with affinity and bioactive domains using genetic engineering. The applicability of herein generated functionalized/bioactivated partial spider silk in disease diagnosis and tissue engineering applications was further investigated. In paper I, affinity domains of different size and fold, which are genetically fused to partial spider silk retain their target binding specificity both when the silk fusion protein is coated onto a solid surface and when processed to fiber format. This means that the affinity domain part of the silk fusion protein is folded in such a way that the binding properties of the added domains are maintained and are exposed on the surface of the silk well enough to enable efficient binding to their target molecules. Growth factors are important cell signaling molecules, which often are indispensable for in vitro cell culture studies. In paper I, the feasibility of a one-step and two-step procedure for non-covalent presentation of growth factors was shown using M4-4RepCT and Z-4RepCT films, respectively. Future potential of M4-4RepCT and Z-4RepCT silk with captured growth factors as in vitro cell culture scaffold should be evaluated. Due to reported high affinity between monomeric streptavidin and biotinylated ligands (Wu & Wong, 2005) release of bound biotinylated ligands usually require harsh elution conditions (Rybak et al., 2004). To allow milder elution of macromolecules captured to M4-4RepCT silk, a biotin analogue reported to bind less tightly, desthiobiotin (Hirsch et al., 2002), could be used instead.

Previously, fibers produced from the 4RepCT protein were reported to be well tolerated when implanted in rats (Fredriksson et al., 2009). Likewise, in future, fibers generated from M4-4RepCT silk could be first used to capture biotinylated antimicrobial drugs, for example, amikacin (Boyce et al., 1993), followed by their implantation to the wounded site. Such type of materials carrying antimicrobial drugs could likely be used for local and sustained delivery of drugs to alleviate wound healing. In paper II, by genetic linkage of single-chain antibody fragments (scFvs) to partial spider silk, it is possible to improve signal strength of immunoassays where the silk fusion protein is spotted onto a array slide, as compared to corresponding array with spots of the antibody fragments alone. The surface properties of the array slide were shown to affect the functionality of immobilized biomolecules (Seurynck-Servoss et al., 2007). Therefore, future study of herein used scFv-silk fusion proteins on various array slides made of different materials could provide better understanding for their efficient use as capture probes in sensitive diagnostic applications. The shelf life of array slides containing capture probes is an important parameter to consider when designing a chip for disease diagnosis. Therefore, long term shelf life studies of the array slides containing scFv-silk fusion proteins should be performed.

Two recombinant antibody fragments (αVEGF and αC1q) used in paper II were selected from a collection of scFvs that contribute to a candidate protein signature for diagnosing the autoimmune disease SLE (Petersson et al., 2014; Carlsson et al., 2011). In future, covalent attachment of other scFvs to the N- terminus of NTCT silk variant could be performed for generation of a sensitive nanoarray slide to diagnose SLE disease. In paper III, bioactivation of partial spider silk with a heparin binding growth factor, basic fibroblast growth factor (bFGF) was achieved via genetic engineering. By combining silk-bFGF with FN-silk, an engineered silk previously developed to support cell adhesion (Widhe et al., 2016), a step toward creation of artificial ECM was verified. The future relevance of herein developed silk based cell scaffold in tissue engineering was first verified by successful in vitro cell culture studies that were performed in medium containing low serum and no added soluble growth factors. In a recent study, a domain in placenta growth factor-2 was reported to bind strongly and promiscuously to ECM proteins (Martino et al., 2014). In future, fusion of genes encoding for such domain and a growth factor to partial spider silk should be investigated for its efficient use in tissue engineering. In situations

when fusion of dimeric and/or glycosylated growth factors to partial spider silk is needed, an immobilization strategy at protein level e.g. sortase mediated ligation could be considered. In vivo, cells use matrix metalloproteinases (MMPs) for degradation and remodeling of the natural ECM (Patterson et al., 2010). Therefore, future incorporation of a MMP recognition site between silk and bFGF parts could be considered for the release of bFGF. Maintenance of pluripotent stem cells in the undifferentiated state requires daily change of media supplemented with soluble bFGF (Lotz et al., 2013). The possibility to culture pluripotent stem cells on combined silk-bFGF and FN-silk coatings in medium without supplemented growth factors should be considered. Furthermore, stem cells cultured on such combined silk coatings should be stained for expression of pluripotent markers. In paper IV, immobilization of two affibody-based binders for VEGFR2 signaling was accomplished by gene fusion to partial spider silk. Fibrils of such silk fusion proteins supported the formation of vessel-like structures within a co-culture of fibroblasts and endothelial cells in a silk scaffold. Expression of VEGF on endothelial and stromal cells can be stimulated by bFGF. The combination of VEGF and bFGF could synergetically increase endothelial cell migration and proliferation (Ito, 2011). As stated in paper IV,

Ztetramer-silk demonstrated agonistic effect for VEGFR2 signaling. Therefore, the combination of Ztetramer-silk and silk-bFGF should be considered for improved performance of endothelial cells. Taken together, the results presented in this thesis highlight the potential of partial spider silk functionalized with affinity domains (e.g. scFvs) for improvement of analyte detection signal in micro- and nanoarrays. Bioactivation of partial spider with cell responsive macromolecules encouraged the future use of silk based cell scaffolds in tissue engineering applications.

4 Popular scientific summary

In the recent times, spider silk has been popular for its amazing mechanical properties in terms of toughness, strength and elasticity. Spider silk is reported to be stronger than steel and high performance synthetic fibers, like Kevlar, by weight. Besides amazing mechanical properties, biocompatibility and biodegradability exhibited by spider silk materials have made them as a promising material for biomedical applications. Certain spiders, e.g. orb weaving spiders, can produce several types of silk from various silk glands and the produced silks vary in their primary amino acid sequence, physical properties and functions. Among several types of silk produced by spiders, dragline silk is the strongest and is used to capture their prey. Researchers have succeeded in deciphering the valuable secrets shared by spiders. However, still there exist no possible way to produce spider silk in commercial quantities. The cannibalistic behavior exhibited by spiders restricted their large scale production. A considerable effort by researchers had resulted in generation of partial spider silk protein in amounts sufficient for certain biomedical applications. Furthermore, compared to most of synthetic materials, partial spider silk is produced using renewable resources. Primarily for economic reasons, E. coli bacteria has often been selected for the production of partial spider silk. Since the large gene size of full length spider silk cannot be handled by bacteria, pieces of the full length spider silk gene have been selected and transferred to bacteria, thus resulting in the production of partial spider silk proteins. The applicability of produced partial spider silk proteins for applications within the field of medicine and biotechnology can be expanded by a process called functionalization, in which different biological properties are incorporated to partial spider silk. Likewise, this thesis is focused on

functionalization of two partial spider silks (4RepCT and NTCT) which has been functionalized with different affinity and bioactive domains using genetic engineering. 4RepCT and NTCT partial spider silks are derived from the dragline silk of the spider E. australis. We have shown that different affinity properties can be incorporated to 4RepCT (in paper I) and NTCT (in paper II) by combining them with different affinity domains, which are useful for different biotechnological applications. In papers III and IV, we have shown that different bioactive domains can be included to 4RepCT. In the silk fusion proteins presented in this thesis, we observed the maintained ability of the 4RepCT (in paper I, III and IV) and NTCT (in paper II) parts to form fibers even after fusion with different affinity and bioactive domains. Furthermore, the target binding properties of the added domains are maintained in all silk fusion proteins investigated (paper I-IV). In paper I, two different ways to present growth factors that are often important for human cell culture was verified using M4-4RepCT and Z- 4RepCT silk materials. Furthermore, the ability to present an active enzyme via M4-4RepCT was investigated. In papers III and IV, we could show the feasibility to bioactivate 4RepCT with growth factors and mimics of growth factor, respectively. Thus, silk materials generated in papers I, III and IV, have been considered for their use in tissue engineering applications. In paper II, two antibody fragments, chosen from a collection of antibody fragments that helps in diagnosing a disease, are genetically attached to 4RepCT and NTCT silk proteins. We have shown the improved target recognition ability of antibody fragments when attached to silk proteins. This suggests the applicability of such functionalized silk proteins for applications where improvement of target recognition signal is needed. Taken together, the applicability of herein developed functionalized partial spider silk materials can be considered for disease diagnosis (paper II) and tissue engineering applications (paper I, III and IV).

Acknowledgements

The work presented in this thesis would not have been possible without the support from following wonderful people.

First and foremost, I would like to express my sincere gratitude and heartfelt thanks to my supervisor My Hedhammar for giving me an opportunity to be a part of her amazing silk group. Thank you very much for providing constant support and encouragement to conduct research over these years. Being part of the silk group is an invaluable learning experience for me both scientifically and personally. I would also like to acknowledge the timely feedback provided on projects, manuscripts and thesis text, which made this thesis possible.

I would like to thank my co-supervisor Christofer Lendel for the nice meetings and for providing comments on the thesis text. Special thanks to Anna Herland for reviewing and providing comments on the thesis text.

Ronnie Jansson, it is really my pleasure to know you from the day you introduced me to AFB department at BMC. Your organizational skills and dedication for research have inspired me to a great extent. Thank you very much for the interesting scientific and general discussions we have had over all these years. Thank you very much for providing comments on section 1.6. Thanks for the nice Table Tennis games. May be now it is time to play a new game? I would like to thank Mona Widhe for being an admirable person and for being highly helpful in discussing lab related topics, mainly related to cell

57 culture. Thank you very much for providing comments on section 1.7 and 1.8, and for her contribution to paper III. Special thanks to Linnea Nilebäck for the nice scientific conversations we have had over these years and for her contribution to paper III. Thanks for the nice time at Kyoto conference. All the best with your PhD work!

I would also like to thank Selina for providing practical help with cell cultures during her stay in the lab. I am grateful to other fantastic members of the silk group as well, Christos, Rajeev, Shashank, Tarek G, Ebba, Elizabeth for their contributions to amazing working environment in the lab. Thanks to the past members of the silk group, Ulrika and Nancy. Special thanks to Adam and Hawraa for very good work and nice company during their stay in the lab.

I would like to highly acknowledge Spiber Technologies AB for providing silk proteins. Very big thanks to the production team, Ingrid Schenning and Wojtek for producing silk proteins whenever needed. I would like to thank present and past members at Spiber Technologies AB for the delicious fikas and nice meetings.

Thank you to all PI’s for constantly maintaining fantastic and stimulating working environment at Plan 3. I feel it is my pleasure to share the office space with nice and wonderful people over these years, including Ronnie, Christiane, Wen, Chris, Sebastian, Johan N, Kristina W, Niklas, Filippa, Andreas J and Feifan. Thank you all for constantly providing friendly environment in the office! I want to thank everyone at Plan 3 for their contribution to the stimulating and inspiring working environment and also for their help over these years. Truly amazing people to work with!

Thanks to Christer, Payam and Linn for their contributions to paper II and special thanks to Payam for his help in the lab during my visit to Lund. Special thanks to Rezan and John, for their contributions to paper IV and for providing timely comments. This is highly appreciated! Many thanks to other collaborators: Dimple Chouhan and Biman Mandal.

I would also like to thank all co-authors of the papers that are included in this thesis.

Thank you very much to VINNOVA for funding this research. Thank you very much to Gålöstiftelsen for providing travel grants that enabled me to attend and present my research work at different International scientific conferences.

During my stay in Sweden so far, I feel navigating the language and cultural barriers is an enjoyable adventure. Over these years, my friends list has grown and became too numerous to acknowledge everyone by name. Therefore, I would like to thank all my Indian friends who are scattered across Uppsala, Stockholm and a few in Gothenburg. A very big thanks to all these people and other people who have made my stay in Sweden as a memorable and enjoyable journey.

I would like to specially thank my friends in India, Sriram, Abhishek and Srikanth who are helpful and supportive whenever needed.

Specially, I would like to express my deepest gratitude and heartfelt thanks to my mother Lakshmigaru and my father Rameshgaru for their unwavering belief in me, unconditional trust, timely encouragement, endless love and support throughout this journey. Simple thanks are not enough to convey my deep respect and gratitude towards both of you. I would like to express my gratitude to my mother-in-law Nagamanigaru and my father-in-law Ramakrishnagaru for their support in this journey. I also want to thank my little sister Namratha, my brother-in-law Dr.Srumith and my other relatives for their encouragement in this journey.

Last but not least, I want to thank with love, my wife Dr.Soundarya for her understanding. She has been my best friend, great companion who has constantly encouraged me in this journey. I am highly grateful to her love and constant support.

References

Affinomics Binder types: classical, recombinant and alternatives. Available at: http://www.affinomics.org/?q=background/binder_types. Agrawal, C.M. & Athanasiou, K.A. (1997). Technique to control pH in vicinity of biodegrading PLA-PGA implants. J Biomed Mater Res, 38(2), pp. 105-14. Akter, F. (2016). Chapter 1 - What is Tissue Engineering? In: Akter, F. (ed. Tissue Engineering Made Easy Academic Press, pp. 1-2. Available from: http://www.sciencedirect.com/science/article/pii/B9780128053614000011 . Altman, G.H., Diaz, F., Jakuba, C., Calabro, T., Horan, R.L., Chen, J., Lu, H., Richmond, J. & Kaplan, D.L. (2003). Silk-based biomaterials. Biomaterials, 24(3), pp. 401-16. Ambrosio, A.M., Sahota, J.S., Khan, Y. & Laurencin, C.T. (2001). A novel amorphous calcium phosphate polymer ceramic for bone repair: I. Synthesis and characterization. J Biomed Mater Res, 58(3), pp. 295-301. An, B., Jenkins, J.E., Sampath, S., Holland, G.P., Hinman, M., Yarger, J.L. & Lewis, R. (2012). Reproducing natural spider silks' copolymer behavior in synthetic silk mimics. Biomacromolecules, 13(12), pp. 3938-48. Aramwit, P., Motta, A. & Kundu, S.C. (2017). Tissue Engineering: From Basic Sciences to Clinical Perspectives. Biomed Res Int, 2017, p. 2. Arcidiacono, S., Mello, C., Kaplan, D., Cheley, S. & Bayley, H. (1998). Purification and characterization of recombinant spider silk expressed in Escherichia coli. Appl Microbiol Biotechnol, 49(1), pp. 31-8. Askarieh, G., Hedhammar, M., Nordling, K., Saenz, A., Casals, C., Rising, A., Johansson, J. & Knight, S.D. (2010). Self-assembly of spider silk proteins is controlled by a pH-sensitive relay. Nature, 465(7295), pp. 236-8. Ayoub, N.A., Garb, J.E., Tinghitella, R.M., Collin, M.A. & Hayashi, C.Y. (2007). Blueprint for a high-performance biomaterial: full-length spider dragline silk genes. PLoS One, 2(6), p. e514. Babensee, J.E., Anderson, J.M., McIntire, L.V. & Mikos, A.G. (1998). Host response to tissue engineered devices. Advanced Drug Delivery Reviews, 33(1), pp. 111-139.

Badylak, S., Gilbert, T. & Myers-Irvin, J. (2008). Chapter 5 - The extracellular matrix as a biologic scaffold for tissue engineering. In: Blitterswijk, C.v., Thomsen, P., Lindahl, A., Hubbell, J., Williams, D.F., Cancedda, R., Bruijn, J.D.d. & Sohier, J. (eds) Tissue Eng. Burlington: Academic Press, pp. 121-143. Available from: http://www.sciencedirect.com/science/article/pii/B9780123708694000057 . Baldwin, J., Antille, M., Bonda, U., De-Juan-Pardo, E.M., Khosrotehrani, K., Ivanovski, S., Petcu, E.B. & Hutmacher, D.W. (2014). In vitro pre- vascularisation of tissue-engineered constructs A co-culture perspective. Vasc Cell, 6, p. 13. Bhat, S. & Kumar, A. (2013). Biomaterials and bioengineering tomorrow's healthcare. Biomatter, 3(3). Bini, E., Foo, C.W., Huang, J., Karageorgiou, V., Kitchel, B. & Kaplan, D.L. (2006). RGD-functionalized bioengineered spider dragline silk biomaterial. Biomacromolecules, 7(11), pp. 3139-45. Bird, R.E., Hardman, K.D., Jacobson, J.W., Johnson, S., Kaufman, B.M., Lee, S.M., Lee, T., Pope, S.H., Riordan, G.S. & Whitlow, M. (1988). Single- chain antigen-binding proteins. Science, 242(4877), pp. 423-6. Bittencourt, D., Oliveira, P.F., Prosdocimi, F. & Rech, E.L. (2012). Protein families, natural history and biotechnological aspects of spider silk. Genet Mol Res, 11(3), pp. 2360-80. Borkner, C.B., Elsner, M.B. & Scheibel, T. (2014). Coatings and films made of silk proteins. ACS Appl Mater Interfaces, 6(18), pp. 15611-25. Borrebaeck, C.A. & Wingren, C. (2009). Design of high-density antibody microarrays for disease proteomics: key technological issues. J Proteomics, 72(6), pp. 928-35. Borrebaeck, C.A.K. & Wingren, C. (2011). Recombinant Antibodies for the Generation of Antibody Arrays. In: Korf, U. (ed. Protein Microarrays: Methods and Protocols. Totowa, NJ: Humana Press, pp. 247-262. Available from: https://doi.org/10.1007/978-1-61779-286-1_17. Boström, T., Nilvebrant, J. & Hober, S. (2012). Purification Systems Based on Bacterial Surface Proteins, 1. Bourzac, K. (2015). Spiders: Web of intrigue. Nature, 519, p. S4. Boyce, S.T., Supp, A.P., Warden, G.D. & Holder, I.A. (1993). Attachment of an aminoglycoside, amikacin, to implantable collagen for local delivery in wounds. Antimicrob Agents Chemother, 37(9), pp. 1890-5. Briquez, P.S., Hubbell, J.A. & Martino, M.M. (2015). Extracellular Matrix- Inspired Growth Factor Delivery Systems for Skin Wound Healing. Adv Wound Care (New Rochelle), 4(8), pp. 479-489. Butler, J., Ni, L., Brown, W., Joshi, K., Chang, J., Rosenberg, B. & Voss, E. (1993). The immunochemistry of sandwich elisas—VI. Greater than 90% of monoclonal and 75% of polyclonal anti-fluorescyl capture antibodies (CAbs) are denatured by passive adsorption. Molecular immunology, 30(13), pp. 1165-1175.

Camarero, J.A. (2008). Recent developments in the site‐specific immobilization of proteins onto solid supports. Peptide Science, 90(3), pp. 450-458. Carlsson, A., Wuttge, D.M., Ingvarsson, J., Bengtsson, A.A., Sturfelt, G., Borrebaeck, C.A. & Wingren, C. (2011). Serum protein profiling of systemic lupus erythematosus and systemic sclerosis using recombinant antibody microarrays. Mol Cell Proteomics, 10(5), p. M110 005033. Carmagnola, I., Ranzato, E. & Chiono, V. (2018). 11 - Scaffold functionalization to support a tissue biocompatibility. In: Deng, Y. & Kuiper, J. (eds) Functional 3D Tissue Engineering Scaffolds Woodhead Publishing, pp. 255-277. Available from: http://www.sciencedirect.com/science/article/pii/B9780081009796000112 . Chen, X., Knight, D.P. & Vollrath, F. (2002). Rheological characterization of nephila spidroin solution. Biomacromolecules, 3(4), pp. 644-8. Costa, S., Azevedo, H.S. & Reis, R.L. (2005). 17 Enzyme Immobilization in Biodegradable Polymers for Biomedical Applications. Currie, H.A., Deschaume, O., Naik, R.R., Perry, C.C. & Kaplan, D.L. (2011). Genetically engineered chimeric silk-silver binding proteins. Adv Funct Mater, 21(15), pp. 2889-2895. DeBlois, C., Côté, M.-F. & Doillon, C.J. (1994). Heparin-fibroblast growth factorfibrin complex: in vitro and in vivo applications to collagen-based materials. Biomaterials, 15(9), pp. 665-672. Diamandis, E.P. & Christopoulos, T.K. (1991). The biotin-(strept)avidin system: principles and applications in biotechnology. Clin Chem, 37(5), pp. 625- 36. Dicko, C., Vollrath, F. & Kenney, J.M. (2004). Spider silk protein refolding is controlled by changing pH. Biomacromolecules, 5(3), pp. 704-10. Ekins, R.P. (1989). Multi-analyte immunoassay. J Pharm Biomed Anal, 7(2), pp. 155-68. Ellington, A.D. & Szostak, J.W. (1990). In vitro selection of RNA molecules that bind specific ligands. Nature, 346, p. 818. Fleetwood, F., Guler, R., Gordon, E., Stahl, S., Claesson-Welsh, L. & Lofblom, J. (2016). Novel affinity binders for neutralization of vascular endothelial growth factor (VEGF) signaling. Cell Mol Life Sci, 73(8), pp. 1671-83. Fredriksson, C., Feinstein, R., Nordling, K., Kratz, G., Johansson, J., Huss, F. & Rising, A. (2009). Tissue response to subcutaneously implanted recombinant spider silk: an in vivo study. Materials, 2(4), pp. 1908-1922. Gattazzo, F., Urciuolo, A. & Bonaldo, P. (2014). Extracellular matrix: a dynamic microenvironment for stem cell niche. Biochim Biophys Acta, 1840(8), pp. 2506-19. Gebauer, M. & Skerra, A. (2009). Engineered protein scaffolds as next-generation antibody therapeutics. Curr Opin Chem Biol, 13(3), pp. 245-55. Gomes, M., Azevedo, H., Malafaya, P., Silva, S., Oliveira, J., Silva, G., João Mano, R.S. & Reis, R. (2013). 16 - Natural Polymers in Tissue Engineering Applications. In: Ebnesajjad, S. (ed. Handbook of

Biopolymers and Biodegradable Plastics. Boston: William Andrew Publishing, pp. 385-425. Available from: http://www.sciencedirect.com/science/article/pii/B9781455728343000161 . Gomes, S.C., Leonor, I.B., Mano, J.F., Reis, R.L. & Kaplan, D.L. (2011). Antimicrobial functionalized genetically engineered spider silk. Biomaterials, 32(18), pp. 4255-66. Gosline, J.M., Guerette, P.A., Ortlepp, C.S. & Savage, K.N. (1999). The mechanical design of spider silks: from fibroin sequence to mechanical function. J Exp Biol, 202(Pt 23), pp. 3295-303. Griffith, L.G. & Naughton, G. (2002). Tissue engineering--current challenges and expanding opportunities. Science, 295(5557), pp. 1009-14. Guarino, V., Benfenati, V., Cruz-Maya, I., Saracino, E., Zamboni, R. & Ambrosio, L. (2018). 2 - Instructive proteins for tissue regeneration. In: Deng, Y. & Kuiper, J. (eds) Functional 3D Tissue Engineering Scaffolds Woodhead Publishing, pp. 23-49. Available from: http://www.sciencedirect.com/science/article/pii/B9780081009796000021 . Guhrs, K.H., Weisshart, K. & Grosse, F. (2000). Lessons from nature--protein fibers. J Biotechnol, 74(2), pp. 121-34. Gupta, S.K., Saxena, P., Pant, V.A. & Pant, A.B. (2012). Release and toxicity of dental resin composite. Toxicol Int, 19(3), pp. 225-34. Hackenberger, C.P. & Schwarzer, D. (2008). Chemoselective ligation and modification strategies for peptides and proteins. Angew Chem Int Ed Engl, 47(52), pp. 10030-74. Hagn, F., Eisoldt, L., Hardy, J.G., Vendrely, C., Coles, M., Scheibel, T. & Kessler, H. (2010). A conserved spider silk domain acts as a molecular switch that controls fibre assembly. Nature, 465(7295), pp. 239-42. Hajimiri, M., Shahverdi, S., Kamalinia, G. & Dinarvand, R. (2015). Growth factor conjugation: strategies and applications. J Biomed Mater Res A, 103(2), pp. 819-38. Hardy, J.G. & Scheibel, T.R. (2009). Silk-inspired polymers and proteins. Biochem Soc Trans, 37(Pt 4), pp. 677-81. Hartmann, M., Roeraade, J., Stoll, D., Templin, M.F. & Joos, T.O. (2009). Protein microarrays for diagnostic assays. Anal Bioanal Chem, 393(5), pp. 1407- 16. Heineken, F.G. & Skalak, R. (1991). Tissue Engineering: A Brief Overview. Journal of Biomechanical Engineering, 113(2), pp. 111-112. Hench, L.L. (1998). Bioceramics. Journal of the American Ceramic Society, 81(7), pp. 1705-1728. Hirsch, J.D., Eslamizar, L., Filanoski, B.J., Malekzadeh, N., Haugland, R.P., Beechem, J.M. & Haugland, R.P. (2002). Easily reversible desthiobiotin binding to streptavidin, avidin, and other biotin-binding proteins: uses for protein labeling, detection, and isolation. Anal Biochem, 308(2), pp. 343- 57.

Holliger, P. & Hudson, P.J. (2005). Engineered antibody fragments and the rise of single domains. Nat Biotechnol, 23(9), pp. 1126-36. Hu, X., Hortiguela, M.J., Robin, S., Lin, H., Li, Y., Moran, A.P., Wang, W. & Wall, J.G. (2013). Covalent and oriented immobilization of scFv antibody fragments via an engineered glycan moiety. Biomacromolecules, 14(1), pp. 153-9. Huang, G., Li, F., Zhao, X., Ma, Y., Li, Y., Lin, M., Jin, G., Lu, T.J., Genin, G.M. & Xu, F. (2017). Functional and Biomimetic Materials for Engineering of the Three-Dimensional Cell Microenvironment. Chemical Reviews, 117(20), pp. 12764-12850. Huang, Z., Salim, T., Brawley, A., Patterson, J., Matthews, K. & Bondos, S. (2011). Functionalization and Patterning of Protein‐Based Materials Using Active Ultrabithorax Chimeras21). Huebsch, N. & Mooney, D.J. (2009). Inspiration and application in the evolution of biomaterials. Nature, 462(7272), pp. 426-32. Humenik, M., Smith, A.M. & Scheibel, T. (2011). Recombinant spider silks— biopolymers with potential for future applications. Polymers, 3(1), pp. 640-661. Hust, M., Jostock, T., Menzel, C., Voedisch, B., Mohr, A., Brenneis, M., Kirsch, M.I., Meier, D. & Dubel, S. (2007). Single chain Fab (scFab) fragment. BMC Biotechnol, 7, p. 14. Hutmacher, D.W. (2000). Scaffolds in tissue engineering bone and cartilage. Biomaterials, 21(24), pp. 2529-43. Hynes, R.O. (2009). The extracellular matrix: not just pretty fibrils. Science, 326(5957), pp. 1216-9. Ito, Y. (2011). 4.416 - Growth Factors and Protein-Modified Surfaces and Interfaces. In: Ducheyne, P. (ed. Comprehensive Biomaterials. Oxford: Elsevier, pp. 247-279. Available from: http://www.sciencedirect.com/science/article/pii/B9780080552941002634 . Jansson, R., Thatikonda, N., Lindberg, D., Rising, A., Johansson, J., Nygren, P.A. & Hedhammar, M. (2014). Recombinant spider silk genetically functionalized with affinity domains. Biomacromolecules, 15(5), pp. 1696-706. Johansson, M.U., Frick, I.M., Nilsson, H., Kraulis, P.J., Hober, S., Jonasson, P., Linhult, M., Nygren, P.A., Uhlen, M., Bjorck, L., Drakenberg, T., Forsen, S. & Wikstrom, M. (2002). Structure, specificity, and mode of interaction for bacterial albumin-binding modules. J Biol Chem, 277(10), pp. 8114- 20. Johansson, U., Ria, M., Avall, K., Dekki Shalaly, N., Zaitsev, S.V., Berggren, P.O. & Hedhammar, M. (2015). Pancreatic Islet Survival and Engraftment Is Promoted by Culture on Functionalized Spider Silk Matrices. PLoS One, 10(6), p. e0130169. Kasoju, N. & Bora, U. (2012). Silk fibroin in tissue engineering. Adv Healthc Mater, 1(4), pp. 393-412.

Khademhosseini, A. & Langer, R. (2016). A decade of progress in tissue engineering. Nat Protoc, 11(10), pp. 1775-81. Kimple, A.J., Muller, R.E., Siderovski, D.P. & Willard, F.S. (2010). A capture coupling method for the covalent immobilization of hexahistidine tagged proteins for surface plasmon resonance. Methods Mol Biol, 627, pp. 91- 100. Knight, D.P. & Vollrath, F. (2001). Changes in element composition along the spinning duct in a Nephila spider. Naturwissenschaften, 88(4), pp. 179-82. Kraulis, P.J., Jonasson, P., Nygren, P.A., Uhlen, M., Jendeberg, L., Nilsson, B. & Kordel, J. (1996). The serum albumin-binding domain of streptococcal protein G is a three-helical bundle: a heteronuclear NMR study. FEBS Lett, 378(2), pp. 190-4. Krishnaji, S.T. & Kaplan, D.L. (2013). Bioengineered chimeric spider silk-uranium binding proteins. Macromol Biosci, 13(2), pp. 256-64. Kumble, K.D. (2003). Protein microarrays: new tools for pharmaceutical development. Anal Bioanal Chem, 377(5), pp. 812-819. Langer, R. & Vacanti, J.P. (1993). Tissue engineering. Science, 260(5110), pp. 920-6. Laschke, M.W., Harder, Y., Amon, M., Martin, I., Farhadi, J., Ring, A., Torio- Padron, N., Schramm, R., Rucker, M., Junker, D., Haufel, J.M., Carvalho, C., Heberer, M., Germann, G., Vollmar, B. & Menger, M.D. (2006). Angiogenesis in tissue engineering: breathing life into constructed tissue substitutes. Tissue Eng, 12(8), pp. 2093-104. Lee, K., Silva, E.A. & Mooney, D.J. (2011). Growth factor delivery-based tissue engineering: general approaches and a review of recent developments. J R Soc Interface, 8(55), pp. 153-70. Lemmon, M.A. & Schlessinger, J. (2010). Cell signaling by receptor tyrosine kinases. Cell, 141(7), pp. 1117-34. Li, Y., Xiao, Y. & Liu, C. (2017). The Horizon of Materiobiology: A Perspective on Material-Guided Cell Behaviors and Tissue Engineering. Chem Rev, 117(5), pp. 4376-4421. Lotz, S., Goderie, S., Tokas, N., Hirsch, S.E., Ahmad, F., Corneo, B., Le, S., Banerjee, A., Kane, R.S., Stern, J.H., Temple, S. & Fasano, C.A. (2013). Sustained levels of FGF2 maintain undifferentiated stem cell cultures with biweekly feeding. PLoS One, 8(2), p. e56289. Lovett, M., Lee, K., Edwards, A. & Kaplan, D.L. (2009). Vascularization strategies for tissue engineering. Tissue Eng Part B Rev, 15(3), pp. 353-70. Luo, T., Mohan, K., Iglesias, P.A. & Robinson, D.N. (2013). Molecular mechanisms of cellular mechanosensing. Nature Materials, 12, p. 1064. Lutolf, M.P. & Hubbell, J.A. (2005). Synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering. Nat Biotechnol, 23(1), pp. 47-55. Marks, J.D., Hoogenboom, H.R., Bonnert, T.P., McCafferty, J., Griffiths, A.D. & Winter, G. (1991). By-passing immunization. Human antibodies from V- gene libraries displayed on phage. J Mol Biol, 222(3), pp. 581-97.

Martino, M.M., Briquez, P.S., Guc, E., Tortelli, F., Kilarski, W.W., Metzger, S., Rice, J.J., Kuhn, G.A., Muller, R., Swartz, M.A. & Hubbell, J.A. (2014). Growth factors engineered for super-affinity to the extracellular matrix enhance tissue healing. Science, 343(6173), pp. 885-8. Massie, C. & Mills, I.G. (2006). The developing role of receptors and adaptors. Nat Rev Cancer, 6(5), pp. 403-9. McGee, G.S., Davidson, J.M., Buckley, A., Sommer, A., Woodward, S.C., Aquino, A.M., Barbour, R. & Demetriou, A.A. (1988). Recombinant basic fibroblast growth factor accelerates wound healing. J Surg Res, 45(1), pp. 145-53. McMillen, P. & Holley, S.A. (2015). Integration of cell-cell and cell-ECM adhesion in vertebrate morphogenesis. Curr Opin Cell Biol, 36, pp. 48-53. Mello, C.M., Soares, J.W., Arcidiacono, S. & Butler, M.M. (2004). Acid extraction and purification of recombinant spider silk proteins. Biomacromolecules, 5(5), pp. 1849-52. Molino, P.J., Higgins, M.J., Innis, P.C., Kapsa, R.M. & Wallace, G.G. (2012). Fibronectin and bovine serum albumin adsorption and conformational dynamics on inherently conducting polymers: a QCM-D study. Langmuir, 28(22), pp. 8433-45. Mulyasasmita, W. & Heilshorn, S.C. (2011). 2.203 - Protein-Engineered Biomaterials: Synthesis and Characterization. In: Ducheyne, P. (ed. Comprehensive Biomaterials. Oxford: Elsevier, pp. 35-52. Available from: http://www.sciencedirect.com/science/article/pii/B9780080552941002555 . Murphy, W.L., McDevitt, T.C. & Engler, A.J. (2014). Materials as stem cell regulators. Nature Materials, 13, p. 547. Murugan, R. & Ramakrishna, S. (2005). Development of nanocomposites for bone grafting. Composites Science and Technology, 65(15), pp. 2385-2406. Nair, L.S. & Laurencin, C.T. (2006). Polymers as Biomaterials for Tissue Engineering and Controlled Drug Delivery. In: Lee, K. & Kaplan, D. (eds) Tissue Engineering I. Berlin, Heidelberg: Springer Berlin Heidelberg, pp. 47-90. Available from: https://doi.org/10.1007/b137240. Netti, P.A. (2014). Chapter 10 - Bioactivated Materials for Cell and Tissue Guidance. In: Orlando, G., Lerut, J., Soker, S. & Stratta, R.J. (eds) Regenerative Medicine Applications in Organ Transplantation. Boston: Academic Press, pp. 137-150. Available from: http://www.sciencedirect.com/science/article/pii/B9780123985231000100 . Nilebäck, L., Hedin, J., Widhe, M., Floderus, L.S., Krona, A., Bysell, H. & Hedhammar, M. (2017). Self-Assembly of Recombinant Silk as a Strategy for Chemical-Free Formation of Bioactive Coatings: A Real-Time Study. Biomacromolecules, 18(3), pp. 846-854.

Nilsson, B., Moks, T., Jansson, B., Abrahmsen, L., Elmblad, A., Holmgren, E., Henrichson, C., Jones, T.A. & Uhlen, M. (1987). A synthetic IgG-binding domain based on staphylococcal protein A. Protein Eng, 1(2), pp. 107-13. Nilvebrant, J. & Hober, S. (2013). The albumin-binding domain as a scaffold for protein engineering. Computational and structural biotechnology journal, 6(7), pp. 1-8. Novosel, E.C., Kleinhans, C. & Kluger, P.J. (2011). Vascularization is the key challenge in tissue engineering. Adv Drug Deliv Rev, 63(4-5), pp. 300-11. Nygren, P.A. (2008). Alternative binding proteins: affibody binding proteins developed from a small three-helix bundle scaffold. Febs j, 275(11), pp. 2668-76. O'Brien, F.J. (2011). Biomaterials & scaffolds for tissue engineering. Materials Today, 14(3), pp. 88-95. Omenetto, F.G. & Kaplan, D.L. (2010). New opportunities for an ancient material. Science, 329(5991), pp. 528-31. Oshige, M., Yumoto, K., Miyata, H., Takahashi, S., Nakada, M., Ito, K., Tamegai, M., Kawaura, H. & Katsura, S. (2013). Immobilization of His-Tagged Proteins on Various Solid Surfaces Using NTA-Modified Chitosan. Patterson, J., Martino, M.M. & Hubbell, J.A. (2010). Biomimetic materials in tissue engineering. Materials Today, 13(1), pp. 14-22. Peppas, N.A. & Khademhosseini, A. (2016). Make better, safer biomaterials. Nature News, 540(7633), p. 335. Petersson, L., Dexlin-Mellby, L., Bengtsson, A.A., Sturfelt, G., Borrebaeck, C.A. & Wingren, C. (2014). Multiplexing of miniaturized planar antibody arrays for serum protein profiling--a biomarker discovery in SLE nephritis. Lab Chip, 14(11), pp. 1931-42. Pollak, A., Blumenfeld, H., Wax, M., Baughn, R.L. & Whitesides, G.M. (1980). Enzyme immobilization by condensation copolymerization into crosslinked polyacrylamide gels. Journal of the American Chemical Society, 102(20), pp. 6324-6336. Popovic, N. & Wilson, E. (2018). 8.04 - Cell Surface Receptors☆. In: McQueen, C.A. (ed. Comprehensive Toxicology (Third Edition). Oxford: Elsevier, pp. 44-54. Available from: http://www.sciencedirect.com/science/article/pii/B9780128012383991748 . Porter, R.R. (1959). The hydrolysis of rabbit y-globulin and antibodies with crystalline papain. Biochem J, 73, pp. 119-26. Pouchkina-Stantcheva, N.N. & McQueen-Mason, S.J. (2004). Molecular studies of a novel dragline silk from a nursery web spider, Euprosthenops sp. (Pisauridae). Comp Biochem Physiol B Biochem Mol Biol, 138(4), pp. 371-6. Prince, J.T., McGrath, K.P., DiGirolamo, C.M. & Kaplan, D.L. (1995). Construction, cloning, and expression of synthetic genes encoding spider dragline silk. Biochemistry, 34(34), pp. 10879-85.

Ren, X., Feng, Y., Guo, J., Wang, H., Li, Q., Yang, J., Hao, X., Lv, J., Ma, N. & Li, W. (2015). Correction: Surface modification and endothelialization of biomaterials as potential scaffolds for vascular tissue engineering applications. Chem Soc Rev, 44(15), p. 5745. Reymond Sutandy, F.X., Qian, J., Chen, C.-S. & Zhu, H. (2013). Overview of Protein Microarrays. Current protocols in protein science / editorial board, John E. Coligan ... [et al.], 0 27, pp. Unit-27.1. Rising, A., Widhe, M., Johansson, J. & Hedhammar, M. (2011). Spider silk proteins: recent advances in recombinant production, structure-function relationships and biomedical applications. Cell Mol Life Sci, 68(2), pp. 169-84. Rockwood, D.N., Preda, R.C., Yucel, T., Wang, X., Lovett, M.L. & Kaplan, D.L. (2011). Materials fabrication from Bombyx mori silk fibroin. Nat Protoc, 6(10), pp. 1612-31. Romer, L. & Scheibel, T. (2008). The elaborate structure of spider silk: structure and function of a natural high performance fiber. Prion, 2(4), pp. 154-61. Rose, F.R., Cyster, L.A., Grant, D.M., Scotchford, C.A., Howdle, S.M. & Shakesheff, K.M. (2004). In vitro assessment of cell penetration into porous hydroxyapatite scaffolds with a central aligned channel. Biomaterials, 25(24), pp. 5507-14. Rouwkema, J., Rivron, N.C. & van Blitterswijk, C.A. (2008). Vascularization in tissue engineering. Trends in Biotechnology, 26(8), pp. 434-441. Rybak, J.N., Scheurer, S.B., Neri, D. & Elia, G. (2004). Purification of biotinylated proteins on streptavidin resin: a protocol for quantitative elution. Proteomics, 4(8), pp. 2296-9. Saerens, D., Huang, L., Bonroy, K. & Muyldermans, S. (2008). Antibody fragments as probe in biosensor development. Sensors, 8(8), pp. 4669- 4686. Sauer-Eriksson, A.E., Kleywegt, G.J., Uhlen, M. & Jones, T.A. (1995). Crystal structure of the C2 fragment of streptococcal protein G in complex with the Fc domain of human IgG. Structure, 3(3), pp. 265-78. Schlessinger, J. (1988). Signal transduction by allosteric receptor oligomerization. Trends in Biochemical Sciences, 13(11), pp. 443-447. Schultz, G.S. & Wysocki, A. (2009). Interactions between extracellular matrix and growth factors in wound healing. Wound Repair Regen, 17(2), pp. 153-62. Schweigerer, L., Neufeld, G., Friedman, J., Abraham, J.A., Fiddes, J.C. & Gospodarowicz, D. (1987). Capillary endothelial cells express basic fibroblast growth factor, a mitogen that promotes their own growth. Nature, 325, p. 257. Scitable, N.e. Available at: https://www.nature.com/scitable/ebooks/cell-biology- for-seminars-14760004/118245291#bookContentViewAreaDivID. Seurynck-Servoss, S.L., White, A.M., Baird, C.L., Rodland, K.D. & Zangar, R.C. (2007). Evaluation of surface chemistries for antibody microarrays. Anal Biochem, 371(1), pp. 105-15.

Shastri, V.P. & Lendlein, A. (2009). Materials in regenerative medicine. Adv Mater, 21(32-33), pp. 3231-4. Shen, M., Rusling, J.F. & Dixit, C.K. (2017). Site-selective orientated immobilization of antibodies and conjugates for immunodiagnostics development. Methods, 116, pp. 95-111. Simmons, A.H., Michal, C.A. & Jelinski, L.W. (1996). Molecular orientation and two-component nature of the crystalline fraction of spider dragline silk. Science, 271(5245), pp. 84-7. Soderlind, E., Strandberg, L., Jirholt, P., Kobayashi, N., Alexeiva, V., Aberg, A.M., Nilsson, A., Jansson, B., Ohlin, M., Wingren, C., Danielsson, L., Carlsson, R. & Borrebaeck, C.A. (2000). Recombining germline-derived CDR sequences for creating diverse single-framework antibody libraries. Nat Biotechnol, 18(8), pp. 852-6. Spieß, K., Wohlrab, S. & Scheibel, T. (2010). Structural characterization and functionalization of engineered spider silk films. Soft Matter, 6(17), pp. 4168-4174. Stark, M., Grip, S., Rising, A., Hedhammar, M., Engstrom, W., Hjalm, G. & Johansson, J. (2007). Macroscopic fibers self-assembled from recombinant miniature spider silk proteins. Biomacromolecules, 8(5), pp. 1695-701. Strimbu, K. & Tavel, J.A. (2010). What are Biomarkers? Current opinion in HIV and AIDS, 5(6), pp. 463-466. Sutherland, T.D., Young, J.H., Weisman, S., Hayashi, C.Y. & Merritt, D.J. (2010). Insect silk: one name, many materials. Annu Rev Entomol, 55, pp. 171-88. Tallawi, M., Rosellini, E., Barbani, N., Cascone, M.G., Rai, R., Saint-Pierre, G. & Boccaccini, A.R. (2015). Strategies for the chemical and biological functionalization of scaffolds for cardiac tissue engineering: a review. J R Soc Interface, 12(108), p. 20150254. Tashiro, M., Tejero, R., Zimmerman, D.E., Celda, B., Nilsson, B. & Montelione, G.T. (1997). High-resolution solution NMR structure of the Z domain of staphylococcal protein A. J Mol Biol, 272(4), pp. 573-90. Thatikonda, N., Nilebäck, L., Kempe, A., Widhe, M. & Hedhammar, M. (2018). Bioactivation of spider silk with basic fibroblast growth factor for in vitro cell culture: a step towards creation of artificial ECM. Tokareva, O., Michalczechen-Lacerda, V.A., Rech, E.L. & Kaplan, D.L. (2013). Recombinant DNA production of spider silk proteins. Microb Biotechnol, 6(6), pp. 651-63. Ullrich, A. & Schlessinger, J. (1990). Signal transduction by receptors with tyrosine kinase activity. Cell, 61(2), pp. 203-212. Vogel, V. & Sheetz, M. (2006). Local force and geometry sensing regulate cell functions. Nat Rev Mol Cell Biol, 7(4), pp. 265-75. Vollrath, F. (2000). Strength and structure of spiders' silks. J Biotechnol, 74(2), pp. 67-83. Wade, R.J. & Burdick, J.A. (2012). Engineering ECM signals into biomaterials. Materials Today, 15(10), pp. 454-459.

Walker, A.A., Weisman, S., Church, J.S., Merritt, D.J., Mudie, S.T. & Sutherland, T.D. (2012). Silk from crickets: a new twist on spinning. PLoS One, 7(2), p. e30408. Wang, X. & Kaplan, D.L. (2011). Functionalization of silk fibroin with NeutrAvidin and biotin. Macromol Biosci, 11(1), pp. 100-10. Wang, Y.J., Shahrokh, Z., Vemuri, S., Eberlein, G., Beylin, I. & Busch, M. (1996). Characterization, stability, and formulations of basic fibroblast growth factor. Pharm Biotechnol, 9, pp. 141-80. Wells, R.G. (2008). The role of matrix stiffness in regulating cell behavior. Hepatology, 47(4), pp. 1394-400. Widhe, M., Bysell, H., Nystedt, S., Schenning, I., Malmsten, M., Johansson, J. & Rising, A. (2010). Recombinant spider silk as matrices for cell culture. Biomaterials, 31(36), pp. 9575-9585. Widhe, M., Johansson, J., Hedhammar, M. & Rising, A. (2012). Invited review current progress and limitations of spider silk for biomedical applications. Biopolymers, 97(6), pp. 468-78. Widhe, M., Johansson, U., Hillerdahl, C.O. & Hedhammar, M. (2013). Recombinant spider silk with cell binding motifs for specific adherence of cells. Biomaterials, 34(33), pp. 8223-34. Widhe, M., Shalaly, N.D. & Hedhammar, M. (2016). A fibronectin mimetic motif improves integrin mediated cell biding to recombinant spider silk matrices. Biomaterials, 74, pp. 256-66. Williams, D.F. (2009). On the nature of biomaterials. Biomaterials, 30(30), pp. 5897-909. Wingren, C. & Borrebaeck, C.A. (2006). Antibody microarrays: current status and key technological advances. Omics, 10(3), pp. 411-27. Wingren, C. & Borrebaeck, C.A. (2007). Progress in miniaturization of protein arrays--a step closer to high-density nanoarrays. Drug Discov Today, 12(19-20), pp. 813-9. Wohlrab, S., Muller, S., Schmidt, A., Neubauer, S., Kessler, H., Leal-Egana, A. & Scheibel, T. (2012). Cell adhesion and proliferation on RGD-modified recombinant spider silk proteins. Biomaterials, 33(28), pp. 6650-9. Worn, A. & Pluckthun, A. (2001). Stability engineering of antibody single-chain Fv fragments. J Mol Biol, 305(5), pp. 989-1010. Wu, S.C. & Wong, S.L. (2005). Engineering soluble monomeric streptavidin with reversible biotin binding capability. J Biol Chem, 280(24), pp. 23225-31. Zeng, X., Shen, Z. & Mernaugh, R. (2012). Recombinant antibodies and their use in biosensors. Anal Bioanal Chem, 402(10), pp. 3027-38. Zhou, L., Lu, G., Shen, L., Wang, L. & Wang, M. (2014). Serum levels of three angiogenic factors in systemic lupus erythematosus and their clinical significance. Biomed Res Int, 2014, p. 627126. Zhu, H. & Qian, J. (2012). Applications of Functional Protein Microarrays in Basic and Clinical Research. Advances in genetics, 79, pp. 123-155.