Optimizing the Affinity and Specificity of Proteins with Molecular Display
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B Cell Activation and Escape of Tolerance Checkpoints: Recent Insights from Studying Autoreactive B Cells
cells Review B Cell Activation and Escape of Tolerance Checkpoints: Recent Insights from Studying Autoreactive B Cells Carlo G. Bonasia 1 , Wayel H. Abdulahad 1,2 , Abraham Rutgers 1, Peter Heeringa 2 and Nicolaas A. Bos 1,* 1 Department of Rheumatology and Clinical Immunology, University Medical Center Groningen, University of Groningen, 9713 Groningen, GZ, The Netherlands; [email protected] (C.G.B.); [email protected] (W.H.A.); [email protected] (A.R.) 2 Department of Pathology and Medical Biology, University Medical Center Groningen, University of Groningen, 9713 Groningen, GZ, The Netherlands; [email protected] * Correspondence: [email protected] Abstract: Autoreactive B cells are key drivers of pathogenic processes in autoimmune diseases by the production of autoantibodies, secretion of cytokines, and presentation of autoantigens to T cells. However, the mechanisms that underlie the development of autoreactive B cells are not well understood. Here, we review recent studies leveraging novel techniques to identify and characterize (auto)antigen-specific B cells. The insights gained from such studies pertaining to the mechanisms involved in the escape of tolerance checkpoints and the activation of autoreactive B cells are discussed. Citation: Bonasia, C.G.; Abdulahad, W.H.; Rutgers, A.; Heeringa, P.; Bos, In addition, we briefly highlight potential therapeutic strategies to target and eliminate autoreactive N.A. B Cell Activation and Escape of B cells in autoimmune diseases. Tolerance Checkpoints: Recent Insights from Studying Autoreactive Keywords: autoimmune diseases; B cells; autoreactive B cells; tolerance B Cells. Cells 2021, 10, 1190. https:// doi.org/10.3390/cells10051190 Academic Editor: Juan Pablo de 1. -
Case of the Anti HIV-1 Antibody, B12
Spectrum of Somatic Hypermutations and Implication on Antibody Function: Case of the anti HIV-1 antibody, b12 Mesfin Mulugeta Gewe A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Washington 2015 Reading Committee: Roland Strong, Chair Nancy Maizels Jessica A. Hamerman Program Authorized to Offer Degree: Molecular and Cellular Biology i ii ©Copyright 2015 Mesfin Mulugeta Gewe iii University of Washington Abstract Spectrum of Somatic Hypermutations and Implication on Antibody Function: Case of the anti HIV-1 antibody, b12 Mesfin Mulugeta Gewe Chair of the Supervisory Committee: Roland Strong, Full Member Fred Hutchinson Cancer Research Center Sequence diversity, ability to evade immune detection and establishment of human immunodeficiency virus type 1 (HIV-1) latent reservoirs present a formidable challenge to the development of an HIV-1 vaccine. Structure based vaccine design stenciled on infection elicited broadly neutralizing antibodies (bNAbs) is a promising approach, in some measure to circumvent existing challenges. Understanding the antibody maturation process and importance of the high frequency mutations observed in anti-HIV-1 broadly neutralizing antibodies are imperative to the success of structure based vaccine immunogen design. Here we report a biochemical and structural characterization for the affinity maturation of infection elicited neutralizing antibodies IgG1 b12 (b12). We investigated the importance of affinity maturation and mutations accumulated therein in overall antibody function and their potential implications to vaccine development. Using a panel of point iv reversions, we examined relevance of individual amino acid mutations acquired during the affinity maturation process to deduce the role of somatic hypermutation in antibody function. -
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. -
Affinity Maturation of a T-Cell Receptor-Like Antibody Specific for A
International Journal of Molecular Sciences Article Affinity Maturation of a T-Cell Receptor-Like Antibody Specific for a Cytomegalovirus pp65-Derived Peptide Presented by HLA-A*02:01 Se-Young Lee 1,†, Deok-Han Ko 1,†, Min-Jeong Son 1, Jeong-Ah Kim 1, Keunok Jung 2 and Yong-Sung Kim 1,2,* 1 Department of Molecular Science and Technology, Ajou University, Suwon 16499, Korea; [email protected] (S.-Y.L.); [email protected] (D.-H.K.); [email protected] (M.-J.S.); [email protected] (J.-A.K.) 2 Department of Allergy and Clinical Immunology, Ajou University School of Medicine, Suwon 16499, Korea; [email protected] * Correspondence: [email protected]; Tel.: +82-31-219-2662; Fax: +82-31-219-1610 † These authors have contributed equally to this work. Abstract: Human cytomegalovirus (CMV) infection is widespread among adults (60–90%) and is usually undetected in healthy individuals without symptoms but can cause severe diseases in im- munocompromised hosts. T-cell receptor (TCR)-like antibodies (Abs), which recognize complex antigens (peptide–MHC complex, pMHC) composed of MHC molecules with embedded short pep- tides derived from intracellular proteins, including pathogenic viral proteins, can serve as diagnostic and/or therapeutic agents. In this study, we aimed to engineer a TCR-like Ab specific for pMHC comprising a CMV pp65 protein-derived peptide (495NLVPMVATV503; hereafter, CMVpp65 ) 495-503 in complex with MHC-I molecule human leukocyte antigen (HLA)-A*02:01 (CMVpp65495-503/HLA- A*02:01) to increase affinity by sequential mutagenesis of complementarity-determining regions Citation: Lee, S.-Y.; Ko, D.-H.; Son, M.-J.; Kim, J.-A.; Jung, K.; Kim, Y.-S. -
What Are the Primary Limitations in B-Cell Affinity Maturation, and How Much Affinity Maturation Can We Drive with Vaccination?: a Role for Antibody Feedback
Downloaded from http://cshperspectives.cshlp.org/ on September 28, 2021 - Published by Cold Spring Harbor Laboratory Press What Are the Primary Limitations in B-Cell Affinity Maturation, and How Much Affinity Maturation Can We Drive with Vaccination? A Role for Antibody Feedback Kai-Michael Toellner,1 Daniel M.-Y. Sze,2 and Yang Zhang1 1Institute of Immunology and Immunotherapy, University of Birmingham, Birmingham B15 2TT, United Kingdom 2School of Health and Biomedical Sciences, RMIT University, Bundoora 3083, Australia Correspondence: [email protected] We discuss the impact of antibody feedback on affinity maturation of B cells. Competition from epitope-specific antibodies produced earlier during the immune response leads to immune complex formation, which is essential for transport and deposition of antigen onto follicular dendritic cells (FDCs). It also reduces the concentration of free epitopes into the mM to nM range, which is essential for B-cell receptors (BCRs) to sense affinity-dependent changes in binding capacity. Antibody feedback may also induce epitope spreading, leading to a broader selection of epitopes recognized by newly emerging B-cell clones. This may be exploitable, providing ways to manipulate epitope usage induced by vaccination. GREAT DEBATES What are the most interesting topics likely to come up over dinner or drinks with your colleagues? Or, more importantly, what are the topics that don’t come up because they are a little too controversial? In Immune Memory and Vaccines: Great Debates, Editors Rafi Ahmed and Shane Crotty have put together a collection of articles on such ques- tions, written by thought leaders in these fields, with the freedom to talk about the issues as they see fit. -
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. -
Vaccine Immunology Claire-Anne Siegrist
2 Vaccine Immunology Claire-Anne Siegrist To generate vaccine-mediated protection is a complex chal- non–antigen-specifc responses possibly leading to allergy, lenge. Currently available vaccines have largely been devel- autoimmunity, or even premature death—are being raised. oped empirically, with little or no understanding of how they Certain “off-targets effects” of vaccines have also been recog- activate the immune system. Their early protective effcacy is nized and call for studies to quantify their impact and identify primarily conferred by the induction of antigen-specifc anti- the mechanisms at play. The objective of this chapter is to bodies (Box 2.1). However, there is more to antibody- extract from the complex and rapidly evolving feld of immu- mediated protection than the peak of vaccine-induced nology the main concepts that are useful to better address antibody titers. The quality of such antibodies (e.g., their these important questions. avidity, specifcity, or neutralizing capacity) has been identi- fed as a determining factor in effcacy. Long-term protection HOW DO VACCINES MEDIATE PROTECTION? requires the persistence of vaccine antibodies above protective thresholds and/or the maintenance of immune memory cells Vaccines protect by inducing effector mechanisms (cells or capable of rapid and effective reactivation with subsequent molecules) capable of rapidly controlling replicating patho- microbial exposure. The determinants of immune memory gens or inactivating their toxic components. Vaccine-induced induction, as well as the relative contribution of persisting immune effectors (Table 2.1) are essentially antibodies— antibodies and of immune memory to protection against spe- produced by B lymphocytes—capable of binding specifcally cifc diseases, are essential parameters of long-term vaccine to a toxin or a pathogen.2 Other potential effectors are cyto- effcacy.