DOCSLIB.ORG

Design and Screening of Degenerate-Codon-Based Protein Ensembles with M13 Bacteriophage

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Design and Screening of Degenerate-Codon-Based Protein Ensembles with M13 Bacteriophage Design and Screening of Degenerate-Codon-Based Protein Ensembles with M13 Bacteriophage by Griffin James Clausen B.S. Biomedical Engineering University of Minnesota – Twin Cities, 2012 Submitted to the Department of Biological Engineering in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Biological Engineering at the Massachusetts Institute of Technology February 2019 © 2019 Massachusetts Institute of Technology. All rights reserved. Signature of Author: ____________________________________________________________ Griffin Clausen Department of Biological Engineering January 14, 2019 Certified by: ___________________________________________________________________ Angela Belcher James Mason Crafts Professor of Biological Engineering and Materials Science Thesis Supervisor Accepted by: ___________________________________________________________________ Forest White Professor of Biological Engineering Graduate Program Committee Chair This doctoral thesis has been examined by the following committee: Amy Keating Thesis Committee Chair Professor of Biology and Biological Engineering Massachusetts Institute of Technology Angela Belcher Thesis Supervisor James Mason Crafts Professor of Biological Engineering and Materials Science Massachusetts Institute of Technology Paul Blainey Core Member, Broad Institute Associate Professor of Biological Engineering Massachusetts Institute of Technology 2 Design and Screening of Degenerate-Codon-Based Protein Ensembles with M13 Bacteriophage by Griffin James Clausen Submitted to the Department of Biological Engineering on January 14, 2019 in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Biological Engineering Abstract A billion years of evolution has crafted a diverse set of proteins capable of complex and varied functionalities. Within recent decades, scientists have applied both rational design and directed evolution to accelerate development of high-value proteins, including biotherapeutics. While computational modelling increasingly facilitates protein design, empirically screening large collections of protein variants remains an essential component of protein engineering. This process requires generating protein variation, partitioning variants with a selection pressure, and identifying highly functional proteins. This thesis presents computational tools for initial protein library design, leverages high-throughput sequencing for phage display screenings, and reports biotemplating of an inorganic phase-change material onto the filamentous M13 phage surface. Designing ensembles of protein variants involves optimizing library size and quality with constraints on screening capacity, cost, and experimental complexity. Incorporating degenerate codons during oligonucleotide synthesis enables residue-specific protein randomization with a known amino acid scope. However, this widely adopted method often generates uneven variant abundances that diverge exponentially with additional randomized residues. The first section of this work presents tools for the design and assessment of degenerate-codon-based protein libraries. This framework facilitates incorporating an arbitrarily large number of randomized sites, non-standard genetic codes, and non-equimolar nucleotide mixtures. In addition to library size and coverage calculations, whole-population diversity metrics and abundance quantiles are reported. An evolutionary solver to optimize non-equimolar base compositions to match amino acid profiles, as well as mutational profiling for spike-in oligonucleotides is also presented. The second section of this thesis develops an experimental and data analysis pipeline for integrating high-throughput DNA sequencing with M13 phage display biopanning. Deeply sequencing naïve M13 peptide libraries elucidated censorship patterns for both p3 and p8 coat protein fusions. Streptavidin panning recapitulated the HPQ binding motif after a single panning round, and additional biopannings pursued M13 p8 variants that interact with both gold films and carbon nanotubes. Furthermore, this thesis explores the effect of M13 p8 surface charge on the biotemplating of an inorganic phase-change material. An ambient temperature synthesis for modulating the atomic composition of germanium-tin-oxide nanomaterials is reported. Thesis Supervisor: Prof. Angela Belcher Title: James Mason Crafts Professor of Biological Engineering and Materials Science 3 Acknowledgements First and foremost, I would like to thank my advisor, Angela Belcher, for her guidance throughout my graduate experience. Angie brings a contagious enthusiasm and creativity to science that can inspire schoolchildren, graduate students, and grey-haired alumni alike. I was drawn to the breadth of research and the ambitious applications pursued by the Belcher Lab, and have learned a tremendous amount over the last few years in this unique environment. Furthermore, I would like to thank my thesis committee members, Amy Keating and Paul Blainey for providing valuable feedback on my graduate research and guidance for my future career. I am very grateful to the members of the Belcher lab for the friendship and assistance generously shared with me. Nimrod, thank you for our countless hours of phage-centric discussion and your second-opinions on perplexing plates. Thank you, Gaelen and Rana, for helping me settle into the lab and launching the deep sequencing project. Thank you to the Bio Subgroup for the illuminating discussions and thank you, Uyanga, for organizing these valuable sessions. Thank you, Eric, for your leadership and willingness to tackle tasks that aim to benefit the broader lab. Thank you, Ngozi, for being a talented community-builder, researcher, reviewer, and ice-cream- cake-shopper. Peter, thank you for the endless conversations. Thank you, Jifa, for your assistance during my 20.109 TA-ship. Thank you, John Casey, for showing me the EHS Rep ropes and thank you, Archana, for taking on the EHS Rep ropes. Thank you, Jackie, Matt, Yu, Nurxat, Xiangnan, Neel, Alan, Will, Swati, George, Briana, Shalmalee, and everyone else! Through my research, I had the pleasure of working with two incredibly hard-working collaborators. Thank you, Professor Desmond Loke, for leading the germanium tin oxide nanowire project as well as our Vermont hiking expedition. Thank you, Dr. Anthony Rojas from the Pentelute Lab, for the expertise and reagents you developed for our exploration of cysteine bioconjugation on phage. I was also fortunate enough to have the assistance of several bright UROPS: Jelle van der Hilst, Devany West, and Pearl Lee. Each of whom made valuable contributions towards the mutagenesis and cloning of phage, peptide conjugation, biopanning experiments, and binding validation assays. While EHS Representative for the lab, I had the chance to engage with many diligent MIT Environment, Health, & Safety employees. Thank you for your guidance Jennifer Lynn, Hans Richter, Wilfred Mbah, Carolyn Stahl, and Roberta Polak. To my fellow Biological Engineering cohort – thank you! Grinding through implementations and exams, with memorable celebrations in Puerto Rico; ski trips and broken wrists; softball and wings; Olympic victories and talent shows; you have left me with many wonderful memories and lasting friendships. And to my excellent 9 Seattle housemates: Santi, Shawn, Kelly, Truby, Frances, Claire, and Khoi- you are such an impressive and fun group of friends that have made my Boston experience unforgettable. Finally, I would like to thank my family. To my parents- thank you for the constant love and support. To my Grandma, thank you for the stream of letters and thoughts. And to Ali, thank you for the endless encouragement and shared excitement for the future. 4 Design and Screening of Degenerate-Codon-Based Protein Ensembles with M13 Bacteriophage Chapters 1 Introduction to M13 Bacteriophage, Protein Libraries, & Ligand Screening 2 Abundance Disparity and Sampling Coverage in Large Degenerate-Oligonucleotide- Based Protein Library Designs 3 High-Throughput Sequencing of Phage-Displayed Libraries and Biopanning Against Biological and Inorganic Targets 4 Biological-Templating of a Segregating Binary Alloy for Nanowire-Like Phase-Change Materials and Memory 5 Summary Appendices A1 General Protocols A2 Supplementary Information for Chapter 2 A3 Supplementary Information for Chapter 3 A4 Supplementary Information for Chapter 4 5 Table of Contents 1 INTRODUCTION TO M13 BACTERIOPHAGE, PROTEIN LIBRARIES, & LIGAND SCREENING Overview ............................................................................................................................. 16 M13 Bacteriophage .............................................................................................................. 17 1.2.1 M13 Genome ............................................................................................................................. 18 1.2.2 M13 Capsid Structure ................................................................................................................ 19 1.2.3 M13 Life Cycle with E. coli Host ................................................................................................. 20 Phage Display and Protein Libraries ...................................................................................... 22 1.3.1 Protein Randomization Techniques........................................................................................... 22 1.3.2 Display Technologies ................................................................................................................
Load more

Recommended publications
  • Wo2015188839a2
    Downloaded from orbit.dtu.dk on: Oct 08, 2021 General detection and isolation of specific cells by binding of labeled molecules Pedersen, Henrik; Jakobsen, Søren; Hadrup, Sine Reker; Bentzen, Amalie Kai; Johansen, Kristoffer Haurum Publication date: 2015 Document Version Publisher's PDF, also known as Version of record Link back to DTU Orbit Citation (APA): Pedersen, H., Jakobsen, S., Hadrup, S. R., Bentzen, A. K., & Johansen, K. H. (2015). General detection and isolation of specific cells by binding of labeled molecules. (Patent No. WO2015188839). General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. Users may download and print one copy of any publication from the public portal for the purpose of private study or research. You may not further distribute the material or use it for any profit-making activity or commercial gain You may freely distribute the URL identifying the publication in the public portal If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. (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 2015/188839
    [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]
  • Modern Methods for Laboratory Diversification of Biomolecules Bratulic and Badran 51
    Available online at www.sciencedirect.com ScienceDirect Modern methods for laboratory diversification of biomolecules Sinisa Bratulic and Ahmed H Badran Genetic variation fuels Darwinian evolution, yet spontaneous to the success of a directed evolution campaign, as the mutation rates are maintained at low levels to ensure cellular sequence landscape of a standard protein or biopolymer is viability. Low mutation rates preclude the exhaustive typically too vast to be exhaustively searched [1]. Where a exploration of sequence space for protein evolution and priori information is limited, unbiased and random in vitro genome engineering applications, prompting scientists to [2,3] or in vivo [4–7] mutagenesis methods have success- develop methods for efficient and targeted diversification of fully generated libraries of variants with improved or nucleic acid sequences. Directed evolution of biomolecules novel functionalities. Alternatively, bioinformatics, struc- relies upon the generation of unbiased genetic diversity to tural, or biochemical information can be leveraged to discover variants with desirable properties, whereas genome- comprehensively explore a portion of the variant land- engineering applications require selective modifications on a scape by focusing mutagenesis on functionally relevant genomic scale with minimal off-targets. Here, we review the sites [8,9]. Finally, newly identified beneficial mutations current toolkit of mutagenesis strategies employed in directed can be isolated or integrated into single sequences using evolution and genome engineering. These state-of-the-art in vitro [10,11] or in vivo [12 ,13] recombination methods. methods enable facile modifications and improvements of Recently, laboratory evolution has shifted to techniques single genes, multicomponent pathways, and whole genomes that directly couple the diversification and assessment for basic and applied research, while simultaneously paving the steps, providing the basis for continuous in vivo evolution way for genome editing therapeutic interventions.
    [Show full text]
  • Combinatorial Reshaping of the Candida Antarctica Lipase a Substrate Pocket for Enantioselectivity Using an Extremely Condensed Library
    Combinatorial reshaping of the Candida antarctica lipase A substrate pocket for enantioselectivity using an extremely condensed library Anders G. Sandström1, Ylva Wikmark1, Karin Engström, Jonas Nyhlén, and Jan-E. Bäckvall2 Department of Organic Chemistry, Arrhenius Laboratory, Stockholm University, SE-106 91 Stockholm, Sweden Edited by Chi-Huey Wong, Academia Sinica, Taipei, Taiwan, and approved October 14, 2011 (received for review July 15, 2011) A highly combinatorial structure-based protein engineering meth- ful in improving properties such as thermostability, substrate od for obtaining enantioselectivity is reported that results in a acceptance or enantioselectivity (17, 18). These methods gener- thorough modification of the substrate binding pocket of Candida ally create small libraries, as only a few amino acid residues are antarctica lipase A (CALA). Nine amino acid residues surrounding targeted. Protein engineering methods that focus on the active the entire pocket were simultaneously mutated, contributing to site has a higher chance of influencing catalytic properties (19). a reshaping of the substrate pocket to give increased enantio- Such active-site-focused libraries have been used with good re- selectivity and activity for a sterically demanding substrate. This sults for the improvement of activity and enantioselectivity (3). approach seems to be powerful for developing enantioselectivity Two or three amino acid positions are generally subjected simul- when a complete reshaping of the active site is required. Screening taneously to mutagenesis to account for the potential synergistic toward ibuprofen ester 1, a substrate for which previously used conformational and electrostatic effects that may appear. This ap- methods had failed, gave variants with a significantly increased proach is called combinatorial active-site saturation test (CAST) enantioselectivity and activity.
    [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]
  • Analyses of All Possible Point Mutations Within a Protein Reveals Relationships Between Function and Experimental Fitness: a Dissertation
    University of Massachusetts Medical School eScholarship@UMMS GSBS Dissertations and Theses Graduate School of Biomedical Sciences 2014-03-25 Analyses of All Possible Point Mutations within a Protein Reveals Relationships between Function and Experimental Fitness: A Dissertation Benjamin P. Roscoe University of Massachusetts Medical School Let us know how access to this document benefits ou.y Follow this and additional works at: https://escholarship.umassmed.edu/gsbs_diss Part of the Biochemistry Commons, Cellular and Molecular Physiology Commons, Molecular Biology Commons, and the Molecular Genetics Commons Repository Citation Roscoe BP. (2014). Analyses of All Possible Point Mutations within a Protein Reveals Relationships between Function and Experimental Fitness: A Dissertation. GSBS Dissertations and Theses. https://doi.org/10.13028/M2G027. Retrieved from https://escholarship.umassmed.edu/gsbs_diss/716 This material is brought to you by eScholarship@UMMS. It has been accepted for inclusion in GSBS Dissertations and Theses by an authorized administrator of eScholarship@UMMS. For more information, please contact [email protected]. ANALYSES OF ALL POSSIBLE POINT MUTATIONS WITHIN A PROTEIN REVEALS RELATIONSHIPS BETWEEN FUNCTION AND EXPERIMENTAL FITNESS A Dissertation Presented By BENJAMIN PETER ROSCOE Submitted to the Faculty of the University of Massachusetts Graduate School of Biomedical Sciences, Worcester in partial fulfillment for the degree of DOCTOR OF PHILOSOPHY (March 25, 2014) BIOCHEMISTRY AND MOLECULAR PHARMACOLOGY ANALYSES OF ALL POSSIBLE POINT MUTATIONS WITHIN A PROTEIN REVEALS RELATIONSHIPS BETWEEN FUNCTION AND EXPERIMENTAL FITNESS A Dissertation Presented By BENJAMIN PETER ROSCOE The signatures of the Dissertation Defense Committee signify completion and approval as to style and content of the Dissertation Daniel N.
    [Show full text]
  • WO 2018/098356 Al 31 May 2018 (31.05.2018) 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 2018/098356 Al 31 May 2018 (31.05.2018) W !P O PCT (51) International Patent Classification: co, California 94124 (US). DUBRIDGE, Robert B.; 825 A61K 39/395 (2006.01) C07K 16/28 (2006.01) Holly Road, Belmont, California 94002 (US). LEMON, A61P 35/00 (2006.01) C07K 16/46 (2006.01) Bryan D.; 2493 Dell Avenue, Mountain View, California 94043 (US). AUSTIN, Richard J.; 1169 Guerrero Street, (21) International Application Number: San Francisco, California 941 10 (US). PCT/US20 17/063 126 (74) Agent: LIN, Clark Y.; WILSON SONSINI GOODRICH (22) International Filing Date: & ROSATI, 650 Page Mill Road, Palo Alto, California 22 November 201 7 (22. 11.201 7) 94304 (US). (25) Filing Language: English (81) Designated States (unless otherwise indicated, for every (26) Publication Langi English kind of national protection available): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ, (30) Priority Data: CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, DO, 62/426,069 23 November 2016 (23. 11.2016) US DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, 62/426,077 23 November 2016 (23. 11.2016) US HR, HU, ID, IL, IN, IR, IS, JO, JP, KE, KG, KH, KN, KP, (71) Applicant: HARPOON THERAPEUTICS, INC. KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, [US/US]; 4000 Shoreline Court, Suite 250, South San Fran MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, cisco, California 94080 (US).
    [Show full text]
  • Methods for the Directed Evolution of Proteins
    REVIEWS STUDY DESIGNS Methods for the directed evolution of proteins Michael S. Packer and David R. Liu Abstract | Directed evolution has proved to be an effective strategy for improving or altering the activity of biomolecules for industrial, research and therapeutic applications. The evolution of proteins in the laboratory requires methods for generating genetic diversity and for identifying protein variants with desired properties. This Review describes some of the tools used to diversify genes, as well as informative examples of screening and selection methods that identify or isolate evolved proteins. We highlight recent cases in which directed evolution generated enzymatic activities and substrate specificities not known to exist in nature. Natural selection Over many generations, iterated mutation and natural In this Review, we summarize techniques that gener- A process by which individuals selection during biological evolution provide solutions ate single-gene libraries, including standard methods as with the highest reproductive for challenges that organisms face in the natural world. well as novel approaches that can generate superior diver- fitness pass on their genetic material to their offspring, thus However, the traits that result from natural selection sity containing a larger proportion of functional mutants. maintaining and enriching only occasionally overlap with features of organ- We also review screening and selection methods that heritable traits that are isms and biomolecules that are sought by humans. identify or isolate improved variants within these librar- adaptive to the natural To guide evolution to access useful phenotypes more ies. Although these strategies can be applied to multi­ environment. frequently, humans for centuries have used artificial gene pathways3,4 and gene networks5–7, the examples in selection Artificial selection , beginning with the selective breeding of this Review will focus exclusively on the laboratory evolu- 1 2 (Also known as selective crops and domestication of animals .
    [Show full text]
  • Nonproteinogenic Deep Mutational Scanning of Linear and Cyclic Peptides
    Nonproteinogenic deep mutational scanning of linear and cyclic peptides Joseph M. Rogersa, Toby Passiouraa, and Hiroaki Sugaa,b,1 aDepartment of Chemistry, Graduate School of Science, The University of Tokyo, Tokyo 113-0033, Japan; and bCore Research for Evolutionary Science and Technology, Japan Science and Technology Agency, Saitama 332-0012, Japan Edited by David Baker, University of Washington, Seattle, WA, and approved September 18, 2018 (received for review June 10, 2018) High-resolution structure–activity analysis of polypeptides re- mutants that can be constructed (18). Moreover, it is possible to quires amino acid structures that are not present in the universal combine parallel peptide synthesis with measures of function genetic code. Examination of peptide and protein interactions (19). However, these approaches cannot construct peptide li- with this resolution has been limited by the need to individually braries with the sequence length and numbers that deep muta- synthesize and test peptides containing nonproteinogenic amino tional scanning can, which, at its core, uses high-fidelity nucleic acids. We describe a method to scan entire peptide sequences with acid-directed synthesis of polypeptides by the ribosome. multiple nonproteinogenic amino acids and, in parallel, determine Ribosomal synthesis (i.e., translation) can be manipulated to the thermodynamics of binding to a partner protein. By coupling include nonproteinogenic amino acids (20). In vitro genetic code genetic code reprogramming to deep mutational scanning, any reprogramming is particularly versatile, allowing for the in- number of amino acids can be exhaustively substituted into pep- corporation of amino acids with diverse chemical structures (21). tides, and single experiments can return all free energy changes of Flexizymes, flexible tRNA-acylation ribozymes, can load almost binding.
    [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]
  • Codongenie: Optimised Ambiguous Codon Design Tools
    A peer-reviewed version of this preprint was published in PeerJ on 10 July 2017. View the peer-reviewed version (peerj.com/articles/cs-120), which is the preferred citable publication unless you specifically need to cite this preprint. Swainston N, Currin A, Green L, Breitling R, Day PJ, Kell DB. 2017. CodonGenie: optimised ambiguous codon design tools. PeerJ Computer Science 3:e120 https://doi.org/10.7717/peerj-cs.120 CodonGenie: optimised ambiguous codon design tools Neil Swainston Corresp., 1 , Andrew Currin 1 , Lucy Green 1 , Rainer Breitling 1, 2 , Philip J Day 3 , Douglas B Kell 1, 2 1 Manchester Centre for Synthetic Biology of Fine and Speciality Chemicals (SYNBIOCHEM), University of Manchester, Manchester, United Kingdom 2 School of Chemistry, University of Manchester, Manchester, United Kingdom 3 Faculty of Biology, Medicine and Health, University of Manchester, Manchester, United Kingdom Corresponding Author: Neil Swainston Email address: [email protected] CodonGenie, freely available from http://codon.synbiochem.co.uk , is a simple web application for designing ambiguous codons to support protein mutagenesis applications. Ambiguous codons are derived from specific heterogeneous nucleotide mixtures, which create sequence degeneracy when synthesised in a DNA library. In directed evolution studies, such codons are carefully selected to encode multiple amino acids. For example, the codon NTN, where the code N denotes a mixture of all four nucleotides, will encode a mixture of phenylalanine, leucine, isoleucine, methionine and valine. Given a user-defined target collection of amino acids matched to an intended host organism, CodonGenie designs and analyses all ambiguous codons that encode the required amino acids.
    [Show full text]
  • WO 2017/156178 Al 14 September 2017 (14.09.2017) 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 2017/156178 Al 14 September 2017 (14.09.2017) P O P C T (51) International Patent Classification: (81) Designated States (unless otherwise indicated, for every C07K 16/28 (2006.01) C07K 16/30 (2006.01) kind of national protection available): AE, AG, AL, AM, C07K 16/18 (2006.01) A61K 39/395 (2006.01) AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, (21) International Application Number: DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, PCT/US2017/021435 HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KH, KN, (22) International Filing Date: KP, KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, 8 March 2017 (08.03.2017) MD, ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, (25) Filing Language: English RU, RW, SA, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, (26) Publication Language: English TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (30) Priority Data: 62/305,092 8 March 2016 (08.03.2016) US (84) Designated States (unless otherwise indicated, for every kind of regional protection available): ARIPO (BW, GH, (71) Applicant: MAVERICK THERAPEUTICS, INC. GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, [US/US]; 3260 B Bayshore Blvd., 1st Floor, Brisbane, CA TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, 94005 (US).
    [Show full text]