DOCSLIB.ORG

Generation of Novel Intracellular Binding Reagents Based on the Human Γb-Crystallin Scaffold

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Generation of Novel Intracellular Binding Reagents Based on the Human Γb-Crystallin Scaffold Generation of novel intracellular binding reagents based on the human γB-crystallin scaffold Dissertation zur Erlangung des akademischen Grades doctor rerum naturalium (Dr. rer. nat.) vorgelegt der Naturwissenschaftlichen Fakultät I-Biowissenschaften der Martin-Luther-Universität Halle-Wittenberg Institut für Biochemie und Biotechnologie von Ewa Mirecka geboren am 17. Dezember 1976 in Gdynia, Polen Table of contents Table of contents 1. INTRODUCTION.........................................................................................................1 1.1 Monoclonal antibodies as a biomolecular scaffold..........................................................1 1.2 Binding molecules derived from non-immunoglobulin scaffolds.....................................3 1.2.1 Alternative protein scaffolds – general considerations............................................................ 3 1.2.2 Application of alternative binding molecules ........................................................................... 6 1.3 Affilin – novel binding molecules based on the human γB-crystallin scaffold................... 6 1.3.1 Human γB-crystallin as a molecular scaffold........................................................................... 6 1.3.2 Generation of a human γB-crystallin library and selection of first-generation Affilin molecules ................................................................................................................................ 8 1.4 Selection of binding proteins by phage display .................................................................... 9 1.5 Human papillomavirus E7 protein as a model target for the generation of intracellular Affilin molecules...................................................................................................................... 11 1.5.1 Human papillomaviruses and associated diseases .............................................................. 12 1.5.2 Genomic organization of human papillomaviruses ............................................................... 12 1.5.3 Viral life cycle......................................................................................................................... 13 1.5.4 Human papillomavirus E7 protein ......................................................................................... 14 1.5.4.1 Biological activities and cellular targets of the E7 protein ............................................. 14 1.5.4.2 Biochemical characterization of the E7 protein ............................................................. 16 1.6 Options for delivery of therapeutic proteins into mammalian cells................................... 18 1.7 Aim of the study ...................................................................................................................... 20 2. MATERIALS AND METHODS ........................................................................ 21 2.1 Materials ................................................................................................................................... 21 2.1.1 Bacterial strains..................................................................................................................... 21 2.1.2 Eukaryotic cells...................................................................................................................... 21 2.1.3 Phage .................................................................................................................................... 21 2.1.4 Vectors .................................................................................................................................. 22 2.1.5 Oligonucleotides.................................................................................................................... 23 2.1.6 Chemicals.............................................................................................................................. 23 2.1.7 Enzymes................................................................................................................................ 24 2.1.8 Proteins ................................................................................................................................. 24 2.1.9 Antibodies.............................................................................................................................. 24 2.1.10 Kits and standards............................................................................................................. 25 2.1.11 Buffers and solutions......................................................................................................... 25 2.1.12 Media and antibiotics for cultivation of E. coli ................................................................... 26 2.1.13 Media for mammalian cell culture...................................................................................... 27 2.1.14 Chromatography columns ................................................................................................. 27 2.1.15 Laboratory equipment ....................................................................................................... 27 2.1.16 Miscellaneous.................................................................................................................... 28 2.2 Methods.................................................................................................................................... 28 2.2.1 Molecular biology................................................................................................................... 28 2.2.1.1 Plasmid DNA preparation.............................................................................................. 28 2.2.1.2 Determination of DNA concentration............................................................................. 28 2.2.1.3 Polymerase chain reaction ............................................................................................ 28 2.2.1.4 DNA digestion................................................................................................................ 29 2.2.1.5 Generation of double-stranded oligonucleotides........................................................... 29 2.2.1.6 Dephosphorylation of DNA............................................................................................ 30 2.2.1.7 Agarose gel electrophoresis.......................................................................................... 30 2.2.1.8 DNA gel extraction......................................................................................................... 30 2.2.1.9 DNA ligation................................................................................................................... 30 2.2.1.10 Site-directed mutagenesis......................................................................................... 30 2.2.1.11 DNA sequencing........................................................................................................ 31 Table of contents 2.2.2 Transformation of E. coli cells ............................................................................................... 31 2.2.2.1 Chemically competent E. coli cells ................................................................................ 31 2.2.2.2 Electrocompetent E. coli cells ....................................................................................... 31 2.2.3 Phage display selection of anti-E7 Affilin molecules............................................................. 32 2.2.4 Protein expression using recombinant E. coli ....................................................................... 35 2.2.5 Protein purification and chromatographic analysis................................................................ 36 2.2.6 Protein characterization......................................................................................................... 37 2.2.6.1 Quantification of protein concentration.......................................................................... 37 2.2.6.2 SDS-PAGE .................................................................................................................... 38 2.2.6.3 Western blotting............................................................................................................. 38 2.2.6.4 ELISA............................................................................................................................. 38 2.2.6.5 Surface plasmon resonance.......................................................................................... 39 2.2.7 Cell biology ............................................................................................................................ 40 2.2.7.1 Mammalian cell propagation ......................................................................................... 40 2.2.7.2 Tat-mediated internalization of Affilin molecules into mammalian cells ........................ 40 2.2.7.3 Transfection of mammalian cells................................................................................... 41 2.2.7.4 Immunofluorescence microscopy.................................................................................. 41 2.2.7.5 Bromodeoxyuridine incorporation assay ....................................................................... 42 2.2.7.6 Analysis of apoptosis..................................................................................................... 42 3. RESULTS .............................................................................................................................
Load more

Recommended publications
  • Fig. 1C Combination Therapy of Tumor Targeted ICOS Agonists with T-Cell Bispecific Molecules
    ( (51) International Patent Classification: (81) Designated States (unless otherwise indicated, for every C07K 16/30 (2006.01) A61P 35/00 (2006.01) kind of national protection av ailable) . AE, AG, AL, AM, C07K 16/28 (2006.01) A 6IK 39/00 (2006.01) AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ, C07K 16/40 (2006.01) CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, (21) International Application Number: HR, HU, ID, IL, IN, IR, IS, JO, JP, KE, KG, KH, KN, KP, PCT/EP20 18/086046 KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, (22) International Filing Date: MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, 20 December 2018 (20. 12.2018) OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, (25) Filing Language: English TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (26) Publication Language: English (84) Designated States (unless otherwise indicated, for every (30) Priority Data: kind of regional protection available) . ARIPO (BW, GH, 17209444.3 2 1 December 2017 (21. 12.2017) EP GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, (71) Applicant (for all designated States except US): F. HOFF- TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, MANN-LA ROCHE AG [CH/CH]; Grenzacherstrasse EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, 124, 4070 Basel (CH).
    [Show full text]
  • Strategies and Challenges for the Next Generation of Therapeutic Antibodies
    FOCUS ON THERAPEUTIC ANTIBODIES PERSPECTIVES ‘validated targets’, either because prior anti- TIMELINE bodies have clearly shown proof of activity in humans (first-generation approved anti- Strategies and challenges for the bodies on the market for clinically validated targets) or because a vast literature exists next generation of therapeutic on the importance of these targets for the disease mechanism in both in vitro and in vivo pharmacological models (experi- antibodies mental validation; although this does not necessarily equate to clinical validation). Alain Beck, Thierry Wurch, Christian Bailly and Nathalie Corvaia Basically, the strategy consists of develop- ing new generations of antibodies specific Abstract | Antibodies and related products are the fastest growing class of for the same antigens but targeting other therapeutic agents. By analysing the regulatory approvals of IgG-based epitopes and/or triggering different mecha- biotherapeutic agents in the past 10 years, we can gain insights into the successful nisms of action (second- or third-generation strategies used by pharmaceutical companies so far to bring innovative drugs to antibodies, as discussed below) or even the market. Many challenges will have to be faced in the next decade to bring specific for the same epitopes but with only one improved property (‘me better’ antibod- more efficient and affordable antibody-based drugs to the clinic. Here, we ies). This validated approach has a high discuss strategies to select the best therapeutic antigen targets, to optimize the probability of success, but there are many structure of IgG antibodies and to design related or new structures with groups working on this class of target pro- additional functions.
    [Show full text]
  • International Patent Classification: KR, KW, KZ, LA, LC, LK, LR, LS, LU
    ( 2 (51) International Patent Classification: DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, A61K 39/42 (2006.01) C07K 16/10 (2006.01) HR, HU, ID, IL, IN, IR, IS, JO, JP, KE, KG, KH, KN, KP, C07K 16/08 (2006.01) KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, (21) International Application Number: OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, PCT/US20 19/033 995 SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, (22) International Filing Date: TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. 24 May 2019 (24.05.2019) (84) Designated States (unless otherwise indicated, for every (25) Filing Language: English kind of regional protection available) . ARIPO (BW, GH, GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ, (26) Publication Language: English UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, (30) Priority Data: TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, 62/676,045 24 May 2018 (24.05.2018) US EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, (71) Applicant: LANKENAU INSTITUTE FOR MEDICAL TR), OAPI (BF, BJ, CF, CG, Cl, CM, GA, GN, GQ, GW, RESEARCH [US/US]; 100 Lancaster Avenue, Wyn- KM, ML, MR, NE, SN, TD, TG).
    [Show full text]
  • WO 2014/152006 A2 25 September 2014 (25.09.2014) P O P C T
    (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2014/152006 A2 25 September 2014 (25.09.2014) P O P C T (51) International Patent Classification: AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, A61K 39/395 (2006.01) BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, (21) International Application Number: HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KN, KP, KR, PCT/US20 14/026804 KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, ME, (22) International Filing Date: MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, 13 March 2014 (13.03.2014) OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, (25) Filing Language: English TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, (26) Publication Language: English ZW. (30) Priority Data: (84) Designated States (unless otherwise indicated, for every 61/791,953 15 March 2013 (15.03.2013) US kind of regional protection available): ARIPO (BW, GH, GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, SZ, TZ, (71) Applicant: INTRINSIC LIFESCIENCES, LLC UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, [US/US]; 505 Coast Boulevard South, Suite 408, La Jolla, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, California 92037 (US).
    [Show full text]
  • WO 2018/144999 Al 09 August 2018 (09.08.2018) W ! P O PCT
    (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2018/144999 Al 09 August 2018 (09.08.2018) W ! P O PCT (51) International Patent Classification: Lennart; c/o Orionis Biosciences NV, Rijvisschestraat 120, A61K 38/00 (2006.01) C07K 14/555 (2006.01) Zwijnaarde, B-9052 (BE). TAVERNIER, Jan; c/o Orionis A61K 38/21 (2006.01) C12N 15/09 (2006.01) Biosciences NV, Rijvisschestraat 120, Zwijnaarde, B-9052 C07K 14/52 (2006.01) (BE). (21) International Application Number: (74) Agent: ALTIERI, Stephen, L. et al; Morgan, Lewis & PCT/US2018/016857 Bockius LLP, 1111 Pennsylvania Avenue, NW, Washing ton, D.C. 20004 (US). (22) International Filing Date: 05 February 2018 (05.02.2018) (81) Designated States (unless otherwise indicated, for every kind of national protection available): AE, AG, AL, AM, (25) Filing Language: English AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ, (26) Publication Language: English CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, (30) Priority Data: HR, HU, ID, IL, IN, IR, IS, JO, JP, KE, KG, KH, KN, KP, 62/454,992 06 February 2017 (06.02.2017) US KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, (71) Applicants: ORIONIS BIOSCIENCES, INC. [US/US]; MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, 275 Grove Street, Newton, MA 02466 (US).
    [Show full text]
  • Affinity Chromatography (AC)
    Affinity Chromatography (AC) 1 Affinity Chromatography (AC) • Principles of AC • Main stages in Chromatography • How to prepare Affinity gel - Ligand Immobilization - Spacer arms – Coupling methods – Coupling tips • Types of AC • Elution Conditions • Binding equilibrium, competitive elution, kinetics • Industrial Examples: Protein A/G for Therapeutic proteins • Future Considerations 2 What is affinity chromatography? Affinity chromatography is a technique of liquid chromatography which separates molecules through biospecific interactions. The molecule to be purified is specifically and reversibly adsorbed to a specific ligand The ligand is immobilized to an insoluble support (“matrix”): resin, “chip”, Elisa plate, membrane, Western, IP (immuneprecipitation), etc Introduction of a “spacer arm” between the ligand and the matrix to improve binding Elution of the bound target molecule a) non specific or b) specific elution method 3 What is it used for? Monoclonal and polyclonal antibodies Fusion proteins Enzymes DNA-binding proteins . ANY protein where we have a binding partner!! 4 Designing and preparing an affinity gel Choosing the matrix Designing the ligand - Spacer arms Coupling methods 5 Ligand Immobilization Ligand + Activating agent + Matrix Activated Immobilised matrix ligand 6 Designing the ligand Essential ligand properties: interacts selectively and reversibly with the target Carries groups which can couple it to the matrix without losing its binding activity Available in a pure form 7 Steric considerations & spacer arms Small ligand (<1,000) Risk of steric interference with binding between matrix and target molecule Often need spacer arm but watch out for Spacer arm adsorption to the spacer! 8 Design of spacer arms Alkyl chain Real risk of unspecific interactions between H H spacer and target molecule O O Hydrophilic chain Risk of unspecific interactions greatly O H reduced No coupling reaction will use 100% of the available binding sites.
    [Show full text]
  • EURL ECVAM Recommendation on Non-Animal-Derived Antibodies
    EURL ECVAM Recommendation on Non-Animal-Derived Antibodies EUR 30185 EN Joint Research Centre This publication is a Science for Policy report by the Joint Research Centre (JRC), the European Commission’s science and knowledge service. It aims to provide evidence-based scientific support to the European policymaking process. The scientific output expressed does not imply a policy position of the European Commission. Neither the European Commission nor any person acting on behalf of the Commission is responsible for the use that might be made of this publication. For information on the methodology and quality underlying the data used in this publication for which the source is neither Eurostat nor other Commission services, users should contact the referenced source. EURL ECVAM Recommendations The aim of a EURL ECVAM Recommendation is to provide the views of the EU Reference Laboratory for alternatives to animal testing (EURL ECVAM) on the scientific validity of alternative test methods, to advise on possible applications and implications, and to suggest follow-up activities to promote alternative methods and address knowledge gaps. During the development of its Recommendation, EURL ECVAM typically mandates the EURL ECVAM Scientific Advisory Committee (ESAC) to carry out an independent scientific peer review which is communicated as an ESAC Opinion and Working Group report. In addition, EURL ECVAM consults with other Commission services, EURL ECVAM’s advisory body for Preliminary Assessment of Regulatory Relevance (PARERE), the EURL ECVAM Stakeholder Forum (ESTAF) and with partner organisations of the International Collaboration on Alternative Test Methods (ICATM). Contact information European Commission, Joint Research Centre (JRC), Chemical Safety and Alternative Methods Unit (F3) Address: via E.
    [Show full text]
  • Anticalins Versus Antibodies: Made-To-Order Binding Proteins for Small Molecules Gregory a Weiss * and Henry B Lowman
    Minireview R177 Anticalins versus antibodies: made-to-order binding proteins for small molecules Gregory A Weiss * and Henry B Lowman Engineering proteins to bind small molecules presents a challenge as daunting as Department of Protein Engineering, Genentech, Inc., drug discovery, for both hinge upon our understanding of receptor^ligand 1 DNA Way, South San Francisco, CA 94080, USA molecular recognition. However, powerful techniques from combinatorial *Present address: Department of Chemistry, molecular biology can be used to rapidly select arti¢cial receptors. While University of California, Irvine, CA 92697, USA traditionally researchers have relied upon antibody technologies as a source of new binding proteins, the lipocalin scaffold has recently emerged as an adaptable Correspondence: HB Lowman receptor for small molecule binding. `Anticalins', engineered lipocalin variants, E-mail: [email protected] offer some advantages over traditional antibody technology and illuminate Keywords: Antibody; Anticalin; Small molecule features of molecular recognition between receptors and small molecule ligands. ligand Received: 19 June 2000 Accepted: 10 July 2000 Published: 1 August 2000 Chemistry & Biology 2000, 7:R177^R184 1074-5521 / 00 / $ ^ see front matter ß 2000 Published by Elsevier Science Ltd. PII: S 1 0 7 4 - 5 5 2 1 ( 0 0 ) 0 0 0 1 6 - 8 Introduction of randomly mutated proteins displayed on the surface of The challenge of discovering small molecule ligands for ¢lamentous bacteriophage, viruses capable of infecting protein receptors is well appreciated by, among others, only bacteria (reviewed in [4]). Diversity can be targeted the pharmaceutical industry, which spent the majority of to particular regions of a displayed protein through stan- its $24 billion pharmaceutical research and development dard molecular biology techniques [5], or can be distrib- budget in 1999 on small molecule drug discovery [1].
    [Show full text]
  • WO 2017/013231 Al 26 January 2017 (26.01.2017) P O P C T
    (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization I International Bureau (10) International Publication Number (43) International Publication Date WO 2017/013231 Al 26 January 2017 (26.01.2017) P O P C T (51) International Patent Classification: BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, C07K 14/705 (2006.01) C07K 16/00 (2006.01) DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KN, KP, KR, (21) International Application Number: KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG, PCT/EP20 16/067468 MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, (22) International Filing Date: PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, 2 1 July 20 16 (21 .07.2016) SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (25) Filing Language: English (84) Designated States (unless otherwise indicated, for every (26) Publication Language: English kind of regional protection available): ARIPO (BW, GH, (30) Priority Data: GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, 62/194,882 2 1 July 2015 (21.07.2015) TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, 62/364,414 20 July 2016 (20.07.2016) TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, (72) Inventors; and LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, (71) Applicants : XIANG, Sue D.
    [Show full text]
  • Bispecific Immunomodulatory Antibodies for Cancer Immunotherapy
    Published OnlineFirst June 9, 2021; DOI: 10.1158/1078-0432.CCR-20-3770 CLINICAL CANCER RESEARCH | REVIEW Bispecific Immunomodulatory Antibodies for Cancer Immunotherapy A C Belen Blanco1,2, Carmen Domínguez-Alonso1,2, and Luis Alvarez-Vallina1,2 ABSTRACT ◥ The recent advances in the field of immuno-oncology have here referred to as bispecific immunomodulatory antibodies, dramatically changed the therapeutic strategy against advanced have the potential to improve clinical efficacy and safety profile malignancies. Bispecific antibody-based immunotherapies have and are envisioned as a second wave of cancer immunotherapies. gained momentum in preclinical and clinical investigations Currently, there are more than 50 bispecific antibodies under following the regulatory approval of the T cell–redirecting clinical development for a range of indications, with promising antibody blinatumomab. In this review, we focus on emerging signs of therapeutic activity. We also discuss two approaches for and novel mechanisms of action of bispecific antibodies inter- in vivo secretion, direct gene delivery, and infusion of ex vivo acting with immune cells with at least one of their arms to gene-modified cells, which may become instrumental for the regulate the activity of the immune system by redirecting and/or clinical application of next-generation bispecific immunomod- reactivating effector cells toward tumor cells. These molecules, ulatory antibodies. Introduction of antibodies, such as linear gene fusions, domain-swapping strat- egies, and self-associating peptides and protein domains (Fig. 1B). The past decade has witnessed a number of cancer immunotherapy Multiple technology platforms are available for the design of bsAbs, breakthroughs, all of which involve the modulation of T cell–mediated allowing fine-tuning of binding valence, stoichiometry, size, flexi- immunity.
    [Show full text]
  • Selection of an Artificial Binding Protein Against the Ectodomain of PTH1R
    Selection of an artificial binding protein against the ectodomain of PTH1R Dissertation Zur Erlangung des akademischen Grades doctor rerum naturalium (Dr. rer. nat.) vorgelegt bei der Naturwissenschaftlichen Fakultät I Biowissenschaften der Martin-Luther-Universität Halle-Wittenberg von Qi Zhang geboren am 21.09.1983 in Shaanxi, China Gutachter /in 1. PD. Dr. Hauke Lilie 2. Prof. Dr. Jochen Balbach 3. Prof. Dr. Annette G. Beck-Sickinger Verteidigungsdatum: 14.03.2013 Zusammenfassung In den vergangenen Jahrzehnten fanden mehr als 30 Immunglobuline (IgGs) und deren Derivate Anwendung in der klinischen Praxis. Trotz des großen Erfolgs solcher Antikörper-basierter Medikamente traten auch einige Limitationen auf. Gerüstproteine stellen eine Alternative zu herkömmlichen Antikörpern dar. Sie weisen meist eine hohe thermodynamische Stabilität auf und bestehen aus einer einzelnen Polypeptidkette ohne Disulfidbrücken. Universelle Bindestellen können wie beim humanen Fibronectin III und bei Anticalinen in flexiblen Loop-Regionen erzeugt werden oder auf rigiden Sekundärstrukturelementen, wie im Fall der Affibodies, DARPine und Affiline. In der vorliegenden Arbeit wurde eine Protein-Bibliothek auf Basis des humanen γB-Kristallins, unter Randomisierung von 8 oberflächenexponierten Aminosäuren auf einem β-Faltblatt der N-terminalen Domäne des Proteins, hergestellt. Ein kürzlich entwickeltes Screening-System, das T7-basierte Phagen-Display, wurde zur Durchmusterung der Bibliothek auf potentielle Binder angewandt. Dabei erfolgt die Assemblierung der Protein-präsentierenden Phagenpartikel ohne einen Transportschritt über die Zellmembran hinweg bereits im Cytoplasma von E. coli. G-Protein gekoppelte Rezeptoren (GPCRs) bilden nur schwerlich für Strukturuntersuchungen geeignete, geordnete Kristallstrukturen aus. Kleine, gut lösliche Bindeproteine könnten sie in einer bestimmten Konformation fixieren und so den Anteil an hydrophilen Resten auf der Proteinoberfläche erhöhen.
    [Show full text]
  • Anticalins: a Novel Class of Therapeutic Binding Proteins
    IPT 23 2007 30/8/07 08:54 Page 32 Biotechnology Anticalins: a Novel Class of Therapeutic Binding Proteins Anticalins – engineered human lipocalin proteins – provide a novel class of biopharmaceutical drug candidates with similar binding and recognition properties as antibodies, while offering several fundamental advantages. By Andreas M. Hohlbaum and Arne Skerra of Pieris AG Andreas M. Hohlbaum (Dr. rer. nat.) is Director of Science and Preclinical Development and member of the management of Pieris. He received his training in immunology and a PhD from the University of Konstanz, Germany, and pursued postdoctoral research at Boston University School of Medicine, US, in the areas of apoptosis, inflammation, innate immunity and tumour immunology. He obtained first-hand experience in commercially oriented Discovery Research during his time at the biopharmaceutical company, Dyax (Cambridge, Mass). Dr Hohlbaum joined Pieris as a Strategic Research Scientist, in February 2003 and was promoted to Assistant Director of Preclinical Research in February 2004. Since May 2006, he has directed Pieris’s research and development of proprietary and also partnered Anticalin-based biotherapeutics. Arne Skerra (Dipl.-Ing., Dr. rer. nat.) is Founder of Pieris, Member of its Supervisory Board, and Professor at the Technische Universität München, where he heads the Institute of Biological Chemistry. He holds a Diploma degree in Chemistry from the Technische Universität Darmstadt, and a PhD in Biochemistry from the Ludwig-Maximilians-Universität München. After post- doctoral studies at the MRC Laboratory of Molecular Biology in Cambridge, UK, and a group leader position at the Max-Planck- Institute for Biophysics in Frankfurt am Main, he became Associate Professor for Protein Chemistry at the TU Darmstadt.
    [Show full text]