Screening of Yeast Display Libraries of Enzymatically Treated Peptides to Discover Macrocyclic Peptide Ligands
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Yeast Surface 2-Hybrid to Detect Protein-Protein Interactions Via the Secretory Pathway As a Platform for Antibody Discovery
CORE Metadata, citation and similar papers at core.ac.uk Provided by Nature Precedings Yeast surface 2-hybrid to detect protein-protein interactions via the secretory pathway as a platform for antibody discovery Xuebo Hu1, Sungkwon Kang1, Xiaoyue Chen1, Charles B Shoemaker2, Moonsoo M Jin1 1Department of Biomedical Engineering, Cornell University, Hungerford Hill Road, Ithaca, NY 14853 2Department of Biomedical Sciences, Tufts Cummings School of Veterinary Medicine, 200 Westboro Rd, North Grafton, MA 01536 Correspondence should be addressed to M.M.J. ([email protected]). Abstract: High throughput methods to measure protein-protein interactions will facilitate uncovering pairs of unknown interactions as well as designing new interactions. We have developed a platform to detect protein interactions on the surface of yeast, where one protein (bait) is covalently anchored to the cell wall and the other (prey) is expressed in secretory form. The prey is released either outside of the cells or remains on the cell surface by its binding to the bait. The strength of their interaction is measured by antibody binding to the epitope tag fused to the prey or direct readout of split fluorescence protein complementation. Our novel ‘yeast surface 2-hybrid’ system was found to differentiate 6-log difference in binding affinities between coiled coil associations and to measure specific interactions of antibodies and antigens. We demonstrate the use of YS2H in exploring activation allostery in integrins and isolating heavy chain only antibodies against botulinum neurotoxin. Introduction Protein-protein interactions are essential to virtually every cellular process and their understanding is of great interest in basic science as well as in the development of effective therapeutics. -
WO 2010/129541 Al
(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 11 November 2010 (11.11.2010) WO 2010/129541 Al (51) International Patent Classification: (81) Designated States (unless otherwise indicated, for every GOlN 33/53 (2006.01) C07K 16/00 (2006.01) kind of national protection available): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, (21) International Application Number: CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO, PCT/US20 10/033537 DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, (22) International Filing Date: HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP, 4 May 2010 (04.05.2010) KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, (25) Filing Language: English NO, NZ, OM, PE, PG, PH, PL, PT, RO, RS, RU, SC, SD, (26) Publication Language: English SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (30) Priority Data: 61/175,076 4 May 2009 (04.05.2009) US (84) Designated States (unless otherwise indicated, for every 61/305,991 19 February 2010 (19.02.2010) US kind of regional protection available): ARIPO (BW, GH, GM, KE, LR, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, (71) Applicant (for all designated States except US): AF- ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, FOMIX CORPORATION [US/US]; 688 E. -
Development of Novel Surface Display Platforms for Anchoring
Yang et al. Microb Cell Fact (2019) 18:85 https://doi.org/10.1186/s12934-019-1133-x Microbial Cell Factories RESEARCH Open Access Development of novel surface display platforms for anchoring heterologous proteins in Saccharomyces cerevisiae Xiaoyu Yang1†, Hongting Tang1,3†, Meihui Song1, Yu Shen1, Jin Hou1* and Xiaoming Bao1,2* Abstract Background: Cell surface display of recombinant proteins has become a powerful tool for biotechnology and biomedical applications. As a model eukaryotic microorganism, Saccharomyces cerevisiae is an ideal candidate for surface display of heterologous proteins. However, the frequently used commercial yeast surface display system, the a-agglutinin anchor system, often results in low display efciency. Results: We initially reconstructed the a-agglutinin system by replacing two anchor proteins with one anchor protein. By directly fusing the target protein to the N-terminus of Aga1p and inserting a fexible linker, the display ef- ciency almost doubled, and the activity of reporter protein α-galactosidase increased by 39%. We also developed new surface display systems. Six glycosylphosphatidylinositol (GPI) anchored cell wall proteins were selected to construct the display systems. Among them, Dan4p and Sed1p showed higher display efciency than the a-agglutinin anchor system. Linkers were also inserted to eliminate the efects of GPI fusion on the activity of the target protein. We further used the newly developed Aga1p, Dan4p systems and Sed1p system to display exoglucanase and a relatively large protein β-glucosidase, and found that Aga1p and Dan4p were more suitable for immobilizing large proteins. Conclusion: Our study developed novel efcient yeast surface display systems, that will be attractive tools for bio- technological and biomedical applications Keywords: Yeast surface display, Aga1, Dan4, Sed1, a-Agglutinin, Glycosylphosphatidylinositol (GPI) Background and antibody development, library screening, biosensor Cell surface display expresses a target protein or peptide detection systems, and bioconversion [2]. -
Peptide Tag Systems That Spontaneously Form An
(19) TZZ ¥_T (11) EP 2 534 484 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: G01N 33/531 (2006.01) C07K 14/315 (2006.01) 19.11.2014 Bulletin 2014/47 C12N 15/00 (2006.01) (21) Application number: 11706621.7 (86) International application number: PCT/GB2011/000188 (22) Date of filing: 11.02.2011 (87) International publication number: WO 2011/098772 (18.08.2011 Gazette 2011/33) (54) PEPTIDE TAG SYSTEMS THAT SPONTANEOUSLY FORM AN IRREVERSIBLE LINK TO PROTEIN PARTNERS VIA ISOPEPTIDE BONDS PEPTIDMARKIERUNGSSYSTEME MIT SPONTANER BILDUNG EINER IRREVERSIBLEN VERBINDUNG ZU PROTEINPARTNERN ÜBER ISOPEPTIDBINDUNGEN SYSTÈMES DE MARQUAGE PEPTIDIQUE QUI FORMENT SPONTANÉMENT UNE LIAISON IRRÉVERSIBLE AVEC DES PARTENAIRES PROTÉIQUES PAR L’INTERMÉDIAIRE DE LIAISONS ISOPEPTIDIQUES (84) Designated Contracting States: • TOMINAGA J ET AL: "Design of a specific peptide AL AT BE BG CH CY CZ DE DK EE ES FI FR GB tagthat affords covalent and site- specificenzyme GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO immobilization catalyzed by microbial PL PT RO RS SE SI SK SM TR transglutaminase", BIOMACROMOLECULES JULY/AUGUST 2005 AMERICAN CHEMICAL (30) Priority: 11.02.2010 GB 201002362 SOCIETY US, vol. 6, no. 4, July 2005 (2005-07), pages 2299-2304, XP002631438, DOI: DOI: (43) Date of publication of application: 10.1021/BM050193O 19.12.2012 Bulletin 2012/51 • DONG RUI-PING ET AL: "Characterization of T cell epitopes restricted by HLA-DP9 in (73) Proprietor: Isis Innovation Limited streptococcal M12 protein", JOURNAL OF Summertown, Oxford OX2 7SQ (GB) IMMUNOLOGY, vol. -
M.Sc. [Botany] 346 13
cover page as mentioned below: below: mentioned Youas arepage instructedcover the to updateupdate to the coverinstructed pageare asYou mentioned below: Increase the font size of the Course Name. Name. 1. IncreaseCourse the theof fontsize sizefont ofthe the CourseIncrease 1. Name. use the following as a header in the Cover Page. Page. Cover 2. the usein the followingheader a as as a headerfollowing the inuse the 2. Cover Page. ALAGAPPAUNIVERSITY UNIVERSITYALAGAPPA [Accredited with ’A+’ Grade by NAAC (CGPA:3.64) in the Third Cycle Cycle Third the in (CGPA:3.64) [AccreditedNAAC by withGrade ’A+’’A+’ Gradewith by NAAC[Accredited (CGPA:3.64) in the Third Cycle and Graded as Category–I University by MHRD-UGC] MHRD-UGC] by University and Category–I Graded as as Graded Category–I and University by MHRD-UGC] M.Sc. [Botany] 003 630 – KARAIKUDIKARAIKUDI – 630 003 346 13 EDUCATION DIRECTORATEDISTANCE OF OF DISTANCEDIRECTORATE EDUCATION BIOLOGICAL TECHNIQUES IN BOTANY I - Semester BOTANY IN TECHNIQUES BIOLOGICAL M.Sc. [Botany] 346 13 cover page as mentioned below: below: mentioned Youas arepage instructedcover the to updateupdate to the coverinstructed pageare asYou mentioned below: Increase the font size of the Course Name. Name. 1. IncreaseCourse the theof fontsize sizefont ofthe the CourseIncrease 1. Name. use the following as a header in the Cover Page. Page. Cover 2. the usein the followingheader a as as a headerfollowing the inuse the 2. Cover Page. ALAGAPPAUNIVERSITY UNIVERSITYALAGAPPA [Accredited with ’A+’ Grade by NAAC (CGPA:3.64) in the Third Cycle Cycle Third the in (CGPA:3.64) [AccreditedNAAC by withGrade ’A+’’A+’ Gradewith by NAAC[Accredited (CGPA:3.64) in the Third Cycle and Graded as Category–I University by MHRD-UGC] MHRD-UGC] by University and Category–I Graded as as Graded Category–I and University by MHRD-UGC] M.Sc. -
Improving Protein Therapeutics Through Quantitative Molecular Engineering Approaches and a Cell-Based Oral Delivery Platform
University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2013 Improving Protein Therapeutics Through Quantitative Molecular Engineering Approaches and A Cell-Based Oral Delivery Platform Ting Wun Ng University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Biomedical Commons Recommended Citation Ng, Ting Wun, "Improving Protein Therapeutics Through Quantitative Molecular Engineering Approaches and A Cell-Based Oral Delivery Platform" (2013). Publicly Accessible Penn Dissertations. 784. https://repository.upenn.edu/edissertations/784 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/784 For more information, please contact [email protected]. Improving Protein Therapeutics Through Quantitative Molecular Engineering Approaches and A Cell-Based Oral Delivery Platform Abstract Proteins, with their ability to perform a variety of highly specific biological functions, have emerged as an important class of therapeutics. However, to fully harness their therapeutic potential, proteins often need to be optimized by molecular engineering; therapeutic efficacy can be improved by modulating protein properties such as binding affinity/specificity, half-life, bioavailability, and immunogenicity. In this work, we first present an introductory example in which a mechanistic mathematical model was used to improve target selection for directed evolution of an aglycosylated Fc domain of an antibody to enhance -
A Bacterial Display System for Effective Selection of Protein-Biotin Ligase Bira Variants with Novel Peptide Specificity
bioRxiv preprint doi: https://doi.org/10.1101/367730; this version posted July 12, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. A bacterial display system for effective selection of protein-biotin ligase BirA variants with novel peptide specificity Jeff Granhøj, Henrik Dimke and Per Svenningsen* Department of Molecular Medicine, University of Southern Denmark, Odense, Denmark * Corresponding author: Dr. Per Svenningsen, Institute of Molecular Medicine, University of Southern Denmark, J.B. Winsloews vej 21.3, DK-5000 Odense C, Denmark. Running title: Bacterial display of enzymatic peptide biotinylation 1 bioRxiv preprint doi: https://doi.org/10.1101/367730; this version posted July 12, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Abstract Biotinylation creates a sensitive and specific tag for purification and detection of target proteins. The E. coli protein-biotin ligase BirA biotinylates a lysine within a synthetic biotin acceptor peptide (AP) and allow for specific tagging of proteins fused to the AP. The approach is not applicable to unmodified proteins, and we sought to develop an effective selection system that could form the basis for directed evolution of novel BirA variants with specificity towards unmodified proteins. The system was based on bacterial display of a target peptide sequence, which could be biotinylated by cytosolic BirA variants before being displayed on the surface. In a model selection, the bacterial display system accomplished >1.000.000 enrichment in a single selection step. -
Supporting Information for a Chemically Synthesized Capture
Supporting Information for A Chemically Synthesized Capture Agent Enables the Selective, Sensitive, and Robust Electrochemical Detection of Anthrax Protective Antigen Blake Farrow 1,2,§ , Sung A Hong 3,§ , Errika C. Romero 2, Bert Lai 4, Matthew B. Coppock 5, Kaycie M. Deyle 2, Amethist S. Finch 5, Dimitra N. Stratis-Cullum 5, Heather D. Agnew 4, Sung Yang 3,* and James R. Heath 2,* 1. Department of Applied Physics and Materials Science, and 2. Division of Chemistry and Chemical Engineering California Institute of Technology 1200 East California Boulevard, Pasadena, California 91125 3. Department of Medical System Engineering Gwangju Institute of Science and Technology Gwangju, 500712, Republic of Korea 4. Indi Molecular 6162 Bristol Parkway, Culver City, California 90230 5. Biotechnology Branch, Sensors & Electronic Devices Directorate U.S. Army Research Laboratory 2800 Powder Mill Rd. Adelphi, MD 20783 § These authors contributed equally to this work. 1 OH (e) NH 2 O NH N NH HN HN O O NH H 2 N O N O H2N NH H O O O O N O O O HN H NH NH N O NH O HN O H O O N O H N O NH 2 O O H O N O NH H N NH O O N O O O O O H2N O C O HN NH H H N O S NH H Figure S1: Screening protocol and anchor ligand. (a-d), Detailed screening steps for identifying biligand capture agent candidates. The details of this protocol are provided in the Supplementary Methods. (b) Structure of anchor ligand used in the biligand screen. -
Chapter 1 Ribosome Display
Chapter 1 Ribosome Display: A Perspective Andreas Plückthun Abstract Ribosome display is an in vitro evolution technology for proteins. It is based on in vitro translation, but prevents the newly synthesized protein and the mRNA encoding it from leaving the ribosome . It thereby couples phenotype and genotype. Since no cells need to be transformed, very large libraries can be used directly in selections, and the in vitro amplifi cation provides a very convenient integration of random mutagenesis that can be incorporated into the procedure. This review highlights concepts, mechanisms, and different variations of ribosome display and compares it to related methods. Applications of ribosome display are summarized, e.g., the directed evolution of proteins for higher binding affi nity, for higher stability or other improved biophysical parameters and enzymatic properties. Ribosome display has developed into a robust technology used in academia and industry alike, and it has made the cell-free Darwinian evolution of proteins over multiple generations a reality. Key words: Directed evolution , Cell-free translation, Ribosome display , Protein engineering, Antibody engineering , DARPins , Designed ankyrin repeat proteins , Affi nity maturation 1. Introduction: Ribosome Display in Context All technologies of molecular evolution must couple phenotype and genotype. There are two fundamental possibilities for achieving this. The fi rst one is compartmentalization. Nature’s compartments are cells: they secure that the superior phenotype expressed by one cell’s mutant genotype can be replicated, without the gene products from the wild type interfering. All selections based on microbial phenotypes use this principle. The second possibility is a direct physical coupling of genetic material to the protein product. -
Antibody Display Technologies: Selecting the Cream of the Crop
Biol. Chem. 2021; aop Review Bernhard Valldorf, Steffen C. Hinz, Giulio Russo, Lukas Pekar, Laura Mohr, Janina Klemm, Achim Doerner, Simon Krah, Michael Hust and Stefan Zielonka* Antibody display technologies: selecting the cream of the crop https://doi.org/10.1515/hsz-2020-0377 other technologies gained a certain level of maturity, most Received November 25, 2020; accepted March 5, 2021; strikingly mammalian display. In this review, we delineate published online March 23, 2021 the most important selection systems with respect to antibody generation with an emphasis on recent Abstract: Antibody display technologies enable the suc- developments. cessful isolation of antigen-specific antibodies with ther- apeutic potential. The key feature that facilitates the Keywords: antibody display; antibody engineering; anti- selection of an antibody with prescribed properties is the body screening; antibody selection; genotype phenotype coupling of the protein variant to its genetic information coupling. and is referred to as genotype phenotype coupling. There are several different platform technologies based on pro- karyotic organisms as well as strategies employing higher Introduction eukaryotes. Among those, phage display is the most established system with more than a dozen of therapeutic Monoclonal antibodies (mAbs) have proven to be remark- antibodies approved for therapy that have been discovered ably versatile therapeutics for disease treatment including or engineered using this approach. In recent years several cancer and inflammatory disorders (Chan and Carter 2010; Scott et al. 2012; Yasunaga, 2020). This is exemplified by Bernhard Valldorf, Steffen C. Hinz, Giulio Russo, and Lukas Pekar the fact that as of November 2019, around 80 mAbs have contributed equally to this work. -
Development of Bacillus Subtilis Spores and Cells for Surface Display of Proteins
Development of Bacillus subtilis spores and cells for surface display of proteins Dissertation zur Erlangung des Grades eines -Doktors der Naturwissenschaften- der Fakultät für Biologie, Chemie und Geowissenschaften der Universität Bayreuth vorgelegt von Nguy ễn Qu ỳnh Anh Bayreuth 2010 Die vorliegende Arbeit wurde in der Zeit von August 2007 bis Oktober 2010 an der Universität Bayreuth am Lehrstuhl für Genetik unter der Betreuung von Prof. Dr. Wolfgang Schumann angefertigt. Vollständiger Abdruck der von der Fakultät für Biologie, Chemie und Geowissenschaften der Universität Bayreuth genehmigten Dissertation zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) Promotionsgesuch eingereicht am: 15.12.2010 Tag des wissenschaftlichen Kolloquiums: 11.03.2011 Erstgutachter: Prof Dr. Wolfgang Schumann Zweitgutachter: PD. Dr. Steffen Kolb Vorsitzender: Prof. Dr. Konrad Dettner Prof. Dr. Franz G. Meussdoerffer Prof. Dr. Birgitta Wöhrl To my parents Acknowledgements First of all, I would like to express my sincere gratitude to my supervisor Prof. Dr. Wolfgang Schumann for his patience, enthusiasm, continuous guidance, and encouragement throughout my research. I also heartily appreciate his substantial support during my years in Bayreuth. I am thankful for Prof. Dr. Thomas Wiegert, whose valuable advices and discussions helped me a lot throughout my working process. Many thanks are due to Prof. Dr. Olaf Stemmann and PD. Dr. Stefan Heidmann for providing facilities for my study. My special thanks also go to Markus Hermann for his help in working with microscope and FACS. My sincere thanks go to Prof. Dr. Junehyung Kim in Dong-A University, Busan, South Korea for his valuable advices and friendly help. -
Understanding Ubiquitin Recognition and Generating Affinity Reagents
Ubiquitin Engineering: Understanding Ubiquitin Recognition and Generating Affinity Reagents by Isabel Leung A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Department of Molecular Genetics University of Toronto © Copyright by Isabel Leung 2017 Abstract Ubiquitin Engineering: Understanding Ubiquitin Recognition and Generating Affinity Reagents Isabel Leung Doctor of Philosophy Department of Molecular Genetics University of Toronto 2016 Protein-protein interactions are necessary for virtually all biological processes. There have been tremendous efforts to document the diversity of molecular recognition, and to understand how molecular recognition occurs. The understanding of molecular interaction has also served as the foundation for designing novel protein interactions for use in therapeutics, diagnostics and basic sciences. An attractive system for studying protein-protein interactions is the ubiquitin (Ub) system. Ub is a protein modifier that is combinatorially ligated onto substrate proteins to influence substrate turnover and function. Ub uses a common surface to interact with more than 1000 proteins and plays pivotal roles in cell physiology. Despite the substantial structural information on Ub mediated interactions, there is no clear understanding of how individual Ub residues contribute to Ub’s broad scope of interactions. To address this question, I used affinity enhanced Ub variants (Ubvs) as proxies of native Ub in saturation scanning. Using saturation scanning, I studied the interactions between Ubvs and two Ub specific proteases (USP), USP2 and USP21, and elucidated a common functional epitope that is critical for USP recognition. The functional epitope recognizes USP residues that are conserved among the human USP family, suggesting it may make functional contributions in many other USP interactions.