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Andreas Pluckthun - One of the best experts on this subject based on the ideXlab platform.
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In-vitro protein evolution by Ribosome Display and mRNA Display.
Journal of immunological methods, 2020Co-Authors: Dasa Lipovsek, Andreas PluckthunAbstract:In-vitro Display technologies combine two important advantages for identifying and optimizing ligands by evolutionary strategies. First, by obviating the need to transform cells in order to generate and select libraries, they allow a much higher library diversity. Second, by including PCR as an integral step in the procedure, they make PCR-based mutagenesis strategies convenient. The resulting iteration between diversification and selection allows true Darwinian protein evolution to occur in vitro. We describe two such selection methods, Ribosome Display and mRNA Display. In Ribosome Display, the translated protein remains connected to the Ribosome and to its encoding mRNA; the resulting ternary complex is used for selection. In mRNA Display, mRNA is first translated and then covalently bonded to the protein it encodes, using puromycin as an adaptor molecule. The covalent mRNA-protein adduct is purified from the Ribosome and used for selection. Successful examples of high-affinity, specific target-binding molecules selected by in-vitro Display methods include peptides, antibodies, enzymes, and engineered scaffolds, such as fibronectin type III domains and synthetic ankyrins, which can mimic antibody function.
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Rapid Selection of High-Affinity Antibody scFv Fragments Using Ribosome Display
Antibody Engineering, 2018Co-Authors: Birgit Dreier, Andreas PluckthunAbstract:Ribosome Display has proven to be a powerful in vitro selection and evolution method for generating high-affinity binders from libraries of folded proteins. It works entirely in vitro, and this has two important consequences. First, since no transformation of any cells is required, libraries with much greater diversity can be handled than with most other techniques. Second, since a library does not have to be cloned and transformed, it is very convenient to introduce random errors in the library by PCR-based methods and select improved binders. Thus, a true directed evolution, an iteration between randomization and selection over several generations, can be conveniently carried out, e.g., for affinity maturation, either on a given clone or on the whole library. Ribosome Display has been successfully applied to antibody single-chain Fv fragments (scFv), which can be selected not only for specificity but also for stability and catalytic activity. High-affinity binders with new target specificity can be obtained from highly diverse libraries in only a few selection rounds. In this protocol, the selection from the library and the process of affinity maturation and off-rate selection are explained in detail.
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Ribosome Display a perspective
Methods of Molecular Biology, 2012Co-Authors: Andreas PluckthunAbstract: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 amplification 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 affinity, 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.
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rapid selection of high affi nity binders using Ribosome Display
2012Co-Authors: Birgit Dreier, Andreas PluckthunAbstract:Ribosome Display has proven to be a powerful in vitro selection and evolution method for generating high-affi nity binders from libraries of folded proteins. It has been successfully applied to single-chain Fv fragments of antibodies and alternative scaffolds, such as D esigned A nkyrin R epeat P roteins (DARPins). High-affi nity binders with new target specifi city can be obtained from highly diverse DARPin libraries in only a few selection rounds. In this protocol, the selection from the library and the process of affi nity maturation and off-rate selection are explained in detail.
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rapid selection of high affinity binders using Ribosome Display
Methods of Molecular Biology, 2012Co-Authors: Birgit Dreier, Andreas PluckthunAbstract:Ribosome Display has proven to be a powerful in vitro selection and evolution method for generating high-affinity binders from libraries of folded proteins. It has been successfully applied to single-chain Fv fragments of antibodies and alternative scaffolds, such as Designed Ankyrin Repeat Proteins (DARPins). High-affinity binders with new target specificity can be obtained from highly diverse DARPin libraries in only a few selection rounds. In this protocol, the selection from the library and the process of affinity maturation and off-rate selection are explained in detail.
Takuya Ueda - One of the best experts on this subject based on the ideXlab platform.
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pure Ribosome Display and its application in antibody technology
Biochimica et Biophysica Acta, 2014Co-Authors: Takashi Kanamori, Yasuhiro Fujino, Takuya UedaAbstract:Abstract Ribosome Display utilizes formation of the mRNA–Ribosome–polypeptide ternary complex in a cell-free protein synthesis system to link genotype (mRNA) to phenotype (polypeptide). However, the presence of intrinsic components, such as nucleases in the cell-extract-based cell-free protein synthesis system, reduces the stability of the ternary complex, which would prevent attainment of reliable results. We have developed an efficient and highly controllable Ribosome Display system using the PURE (Protein synthesis Using Recombinant Elements) system. The mRNA–Ribosome–polypeptide ternary complex is highly stable in the PURE system, and the selected mRNA can be easily recovered because activities of nucleases and other inhibitory factors are very low in the PURE system. We have applied the PURE Ribosome Display to antibody engineering approaches, such as epitope mapping and affinity maturation of antibodies, and obtained results showing that the PURE Ribosome Display is more efficient than the conventional method. We believe that the PURE Ribosome Display can contribute to the development of useful antibodies. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody.
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peptide screening using pure Ribosome Display
Methods of Molecular Biology, 2012Co-Authors: Hiroyuki Ohashi, Takashi Kanamori, Eriko Osada, Bintang K Akbar, Takuya UedaAbstract:To demonstrate directed protein evolution or selection of functional polypeptides, Ribosome Display is one of the most ideal technologies of evolutionary engineering. Intrinsic components, such as nucleases in the cell extract-based cell-free protein synthesis systems, reduce the stability of the messenger RNA-Ribosome-polypeptide ternary complex, thereby preventing the attainment of reliable results. To overcome this problem, we have developed an effective and highly controllable Ribosome Display system using the protein synthesizing using recombinant elements (PURE) system. Since the activities of nucleases and other inhibitory factors are very low in the PURE system, the ternary complex is highly stable and the selected mRNA can be reliably recovered. Using this system, we were able to select peptides that specifically bind to monoclonal antibodies from random peptide libraries. The advantages of the modified PURE system for Ribosome Display strongly substantiate its usability.
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Ribosome Display with the pure technology
Methods of Molecular Biology, 2010Co-Authors: Takuya Ueda, Takashi Kanamori, Hiroyuki OhashiAbstract:The Ribosome Display utilizes the formation of the mRNA-Ribosome-polypeptide ternary complex in the cell-free protein synthesis system as the linking of the genotype (mRNA) to the phenotype (polypeptide). However, the presence of intrinsic components such as nucleases in the cell-extract based cell-free protein synthesis systems inevitably reduces the stability of the ternary complex, which would prevent attainment of reliable results. We have developed an efficient and highly controllable Ribosome Display system using the Protein synthesis Using Recombinant Elements (PURE) system. The mRNA-Ribosome-polypeptide ternary complex is highly stable in the PURE system and then the selected mRNA can be easily recovered, because activities of nucleases and other inhibitory factors are very low in the PURE system. Furthermore, omission of the release factors within the original PURE system can aid stalling of the Ribosome at the termination codon to form the mRNA-Ribosome-polypeptide ternary complex. We believe that these advantages assure the usability of the modified PURE system for Ribosome Display.
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epitope mapping using Ribosome Display in a reconstituted cell free protein synthesis system
Journal of Biochemistry, 2009Co-Authors: Eriko Osada, Takashi Kanamori, Yoshihiro Shimizu, Bintang K Akbar, Takuya UedaAbstract:Ribosome Display is a powerful technology for selecting ligand-binding peptides or proteins. We demonstrate here that the Ribosome Display using the reconstituted cell-free protein synthesis system can be applied for the epitope mapping of monoclonal antibodies (mAbs). Using this technology, we selected peptides that specifically bind to three mAbs from random peptide library. When selection was performed against the anti-FLAG M2 antibody, selected peptides contained previously characterized consensus epitope, indicating that the methodology can be applied for the epitope mapping. When the selection was carried out against two anti-beta-Catenin (anti-beta-Cat) mAbs, selected peptides had a homology for the partial peptide sequences of beta-Cat. Western blot analysis showed that these putative epitopes had affinity for the corresponding mAbs and beta-Cat mutants that lack these regions did not bind to the antibodies, indicating we correctly mapped the epitope for these mAbs. The study shown here provides a way for the quick identification of the epitope of mAbs.
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Epitope Mapping Using Ribosome Display in a Reconstituted Cell-Free Protein Synthesis System
Journal of Biochemistry, 2009Co-Authors: Eriko Osada, Takashi Kanamori, Yoshihiro Shimizu, Bintang K Akbar, Takuya UedaAbstract:Ribosome Display is a powerful technology for selecting ligand-binding peptides or proteins. We demonstrate here that the Ribosome Display using the reconstituted cell-free protein synthesis system can be applied for the epitope mapping of monoclonal antibodies (mAbs). Using this technology, we selected peptides that specifically bind to three mAbs from random peptide library. When selection was performed against the anti-FLAG M2 antibody, selected peptides contained previously characterized consensus epitope, indicating that the methodology can be applied for the epitope mapping. When the selection was carried out against two anti-β-Catenin (anti-P-Cat) mAbs, selected peptides had a homology for the partial peptide sequences of p-Cat. Western blot analysis showed that these putative epitopes had affinity for the corresponding mAbs and β-Cat mutants that lack these regions did not bind to the antibodies, indicating we correctly mapped the epitope for these mAbs. The study shown here provides a way for the quick identification of the epitope of mAbs.
Mingyue He - One of the best experts on this subject based on the ideXlab platform.
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Cell‐free Ribosome Display and selection of antibodies on arrayed antigens
Proteomics, 2016Co-Authors: Cong Cong, Yongxia Zhang, Xin Yu, Yongzhi He, Mingrong Wang, Mingyue HeAbstract:In vitro Display technology is a powerful tool for discovery and optimisation of novel antibodies. With increasing demands on various binding molecules in proteomics studies, techniques for a large-scale generation of antibodies or antibody fragments are needed. Here, we describe a novel method for parallel generation of different antibody fragments (scFv) by integrating cell-free Ribosome Display with array technology. We have demonstrated the procedure by successfully isolating scFv antibodies specific to 16 different cancer biomarkers via a single process. Our results provide proof of principle for multiple production of various scFv antibodies simultaneously.
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cell free Ribosome Display and selection of antibodies on arrayed antigens
Proteomics, 2016Co-Authors: Cong Cong, Yongxia Zhang, Xin Yu, Yongzhi He, Mingrong Wang, Mingyue HeAbstract:In vitro Display technology is a powerful tool for discovery and optimisation of novel antibodies. With increasing demands on various binding molecules in proteomics studies, techniques for a large-scale generation of antibodies or antibody fragments are needed. Here, we describe a novel method for parallel generation of different antibody fragments (scFv) by integrating cell-free Ribosome Display with array technology. We have demonstrated the procedure by successfully isolating scFv antibodies specific to 16 different cancer biomarkers via a single process. Our results provide proof of principle for multiple production of various scFv antibodies simultaneously.
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accessing of recombinant human monoclonal antibodies from patient libraries by eukaryotic Ribosome Display
Human antibodies, 2012Co-Authors: Jie Tang, Lin Wang, Anatoliy Markiv, Simon A Jeffs, Hanna Dreja, Aine Mcknight, Mingyue HeAbstract:What are effective antibodies and when do they arise to prevent or delay disease onset during a natural infection or in the course of vaccination? To address these questions at a molecular level requires longitudinal studies, capturing and analyzing the antibody repertoire at regular intervals following exposure or sero-conversion. Such studies require a method that allows the rapid generation and evaluation of monoclonal antibodies from relatively small volumes of blood. Here we describe an approach for rapidly generating human monoclonal antibodies in vitro by directly screening single-chain antibody repertories derived from donor peripheral blood mononuclear cells using Ribosome Display. Two single-chain antibody libraries were constructed using RNA extracted from peripheral blood mononuclear cells of two HIV-1 long-term non-progressor donors (K530 and M325). Both libraries were subjected to a single round of in vitro Ribosome Display for enrichment of human monoclonal antibodies against recombinant gp120K530, derived from virus isolated from donor K530. This study has validated a novel, in vitro method for the rapid generation of human monoclonal antibodies. An antibody library could be constructed from as little as 3 μg of total RNA, the equivalent of 3–5 mL of human blood.
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Ribosome Display and screening for protein therapeutics
Methods of Molecular Biology, 2012Co-Authors: Damjana Kastelic, Mingyue HeAbstract:Ribosome Display is a cell-free Display technology which enables in vitro selection of antibodies from large recombinant DNA libraries. It also allows continuous introduction of mutations into the selected DNA pool by PCR-based mutagenesis in each cycle, enabling selection of antibody variants with improved affinity, specificity, and stability, thus providing a powerful "protein evolution" tool for optimizing antibody therapeutics. Ribosome Display selects required molecules by linking individual proteins (phenotype) with their corresponding mRNAs (genotype) through the formation of stable Protein-Ribosome-mRNA (PRM) complexes. By affinity interaction with an immobilized ligand, the captured PRM complexes are recovered as cDNA using RT-PCR from the Ribosome-attached mRNA. The DNA is then subjected to subsequent Ribosome Display cycles for further enrichment of rare species or cloning, expression, and sequencing to identify wanted candidates. Both prokaryotic and eukaryotic cell-free systems have been developed for Ribosome Display of different proteins. In this chapter, we describe Ribosome Display of antibodies using the eukaryotic rabbit reticulocyte system with an in situ single-primer DNA recovery method. A high-throughput Escherichia coli expression format is also described for screening of individual antibody binders from the Ribosome-selected population.
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evolution of antibodies in vitro by Ribosome Display
Methods of Molecular Biology, 2012Co-Authors: Bryan M Edwards, Mingyue HeAbstract:Ribosome Display is a cell-free technology which enables in vitro selection and evolution of antibodies from very large diversified DNA libraries. It operates through the following key steps: (1) generation of PCR library; (2) formation of stable antibody-Ribosome-mRNA (ARM) complexes as the selection particles, (3) selection of ligand-binding ARM complexes on an immobilized ligand and (4) recovery of the selected genetic information as DNA by RT-PCR. Since PCR-based random or/and site-directed mutagenesis can be easily used to introduce mutations into the selected DNA pool in each cycle, Ribosome Display offers an efficient "protein evolution" tool for antibody optimization. Both prokaryotic and eukaryotic cell-free systems have been explored for Ribosome Display of different proteins. In this chapter, we describe the use of the eukaryotic rabbit reticulocyte Ribosome Display method to isolate variants of V(H) antibody fragments with improved affinities.
Michael J Taussig - One of the best experts on this subject based on the ideXlab platform.
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eukaryotic Ribosome Display with in situ dna recovery
Methods of Molecular Biology, 2012Co-Authors: Mingyue He, Damjana Kastelic, Bryan M Edwards, Michael J TaussigAbstract:Ribosome Display is a cell-free Display technology for in vitro selection and optimisation of proteins from large diversified libraries. It operates through the formation of stable protein-Ribosome-mRNA (PRM) complexes and selection of ligand-binding proteins, followed by DNA recovery from the selected genetic information. Both prokaryotic and eukaryotic Ribosome Display systems have been developed. In this chapter, we describe the eukaryotic rabbit reticulocyte method in which a distinct in situ single-primer RT-PCR procedure is used to recover DNA from the selected PRM complexes without the need for prior disruption of the Ribosome.
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detection of protein protein interactions by Ribosome Display and protein in situ immobilisation
New Biotechnology, 2009Co-Authors: Mingyue He, Martin R Turner, Michael J TaussigAbstract:We describe a method for identification of protein-protein interactions by combining two cell-free protein technologies, namely Ribosome Display and protein in situ immobilisation. The method requires only PCR fragments as the starting material, the target proteins being made through cell-free protein synthesis, either associated with their encoding mRNA as Ribosome complexes or immobilised on a solid surface. The use of Ribosome complexes allows identification of interacting protein partners from their attached coding mRNA. To demonstrate the procedures, we have employed the lymphocyte signalling proteins Vav1 and Grb2 and confirmed the interaction between Grb2 and the N-terminal SH3 domain of Vav1. The method has promise for library screening of pairwise protein interactions, down to the analytical level of individual domain or motif mapping.
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selection of recombinant antibodies by eukaryotic Ribosome Display
Methods of Molecular Biology, 2008Co-Authors: Mingyue He, Michael J TaussigAbstract:Ribosome Display is a powerful method for selection of single-chain antibodies in vitro. It operates through the formation of libraries of antibody-Ribosome-mRNA complexes that are selected on immobilized antigen, followed by recovery of the genetic information from the mRNA by RT-PCR. Both prokaryotic and eukaryotic versions are used. We describe our eukaryotic system, in which rabbit reticulocyte extracts are used for cell free transcription/translation and cDNA is recovered by in situ RT-PCR performed on the selected complexes.
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selection of antigenic markers on a gfp cκ fusion scaffold with high sensitivity by eukaryotic Ribosome Display
Biochemical and Biophysical Research Communications, 2007Co-Authors: Yongmin Yang, Mingyue He, Michael J Taussig, Teresa J Barankiewicz, Sweyshen ChenAbstract:Abstract Ribosome Display is a cell-free system permitting gene selection through the physical association of genetic material (mRNA) and its phenotypic (protein) product. While often used to select single-chain antibodies from large libraries by panning against immobilized antigens, we have adapted Ribosome Display for use in the ‘reverse’ format in order to select high affinity antigenic determinants against solid-phase antibody. To create an antigenic scaffold, DNA encoding green fluorescent protein (GFP) was fused to a light chain constant domain (Cκ) with stop codon deleted, and with 5′ signals (T7 promoter, Kozak) enabling coupled transcription/translation in a eukaryotic cell-free system. Epitopes on either GFP (5′) or Cκ (3′) were selected by anti-GFP or anti-Cκ antibodies, respectively, coupled to magnetic beads. After selection, mRNA was amplified directly from protein–Ribosome–mRNA (PRM) complexes by in situ PCR followed by internal amplification and reassembly PCR. As little as 10 fg of the 1 kb DNA construct, i.e. approximately 7500 molecules, could be recovered following a single round of interaction with solid-phase anti-GFP antibody. This platform is highly specific and sensitive for the antigen–antibody interaction and may permit selection and reshaping of high affinity antigenic variants of scaffold proteins.
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eukaryotic Ribosome Display with in situ dna recovery
Nature Methods, 2007Co-Authors: Mingyue He, Michael J TaussigAbstract:Ribosome Display is a cell-free technology for the in vitro selection and evolution of proteins encoded by DNA libraries, in which individual nascent proteins (phenotypes) are linked physically to their corresponding mRNA (genotypes) in stable protein-Ribosome-mRNA (PRM) complexes1,2 (Fig. 1). Formation of the complexes can be achieved through deletion of the stop codon of the mRNA, stalling the Ribosome at the end of translation; the nascent protein is extended by a spacer such as the immunoglobulin Cκ domain or others to allow exit through the Ribosome tunnel3. Through affinity for a ligand, the protein-mRNA coupling permits simultaneous isolation of a functional nascent protein and its translated mRNA; the latter is then converted into cDNA by reverse transcription and amplified for further manipulation, repeated cycles or soluble protein expression. Through the use of PCR-generated libraries, avoiding the need for cloning, Ribosome Display can be used to both screen very large populations and continuously search for new diversity during subsequent rounds of selection. Additionally, the use of cell-free systems allows the selection of proteins that are toxic or unstable in cells, and proteins with chemical modifications. Ribosome Display systems using both prokaryotic and eukaryotic cell extracts have been developed1,2. Examples of the application of eukaryotic systems include the selection and evolution of antibody fragments, DNA binding domains, enzymes, interacting proteins and peptides among others3. Here we describe the step-by-step procedure to perform our previously described eukaryotic Ribosome Display method, which has the distinctive feature of an in situ reverse transcription–PCR (RT-PCR) procedure for DNA recovery from Ribosome-bound mRNA1,4,5,6. We also introduce a recent, previously unpublished improvement to the procedure in which in situ reverse transcription is combined with sensitive single-primer PCR technology.
Lutz Jermutus - One of the best experts on this subject based on the ideXlab platform.
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harnessing phage and Ribosome Display for antibody optimisation
Trends in Biotechnology, 2006Co-Authors: Patrick Dufner, Lutz Jermutus, Ralph MinterAbstract:Therapeutic antibodies have become a major driving force for the biopharmaceutical industry; therefore, the discovery and development of safe and efficacious antibody leads have become competitive processes. Phage and Ribosome Display are ideal tools for the generation of such molecules and have already delivered an approved drug as well as a multitude of clinical candidates. Because they are capable of searching billions of antibody variants in tailored combinatorial libraries, they are particularly applicable to potency optimisation. In conjunction with targeted, random or semi-rational mutagenesis strategies, they deliver large panels of potent antibody leads. This review introduces the two technologies, compares them with respect to their use in antibody optimisation and highlights how they can be exploited for the successful and efficient generation of putative drug candidates.
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an improved method for an efficient and easily accessible eukaryotic Ribosome Display technology
Protein Engineering Design & Selection, 2006Co-Authors: Julie A Douthwaite, Maria Groves, P Dufner, Lutz JermutusAbstract:Ribosome Display is a powerful in vitro technology for the selection and directed evolution of proteins. However, this technology has so far been perceived as being technically challenging owing to comparatively difficult protocols and the absence of tailored commercial reagents, particularly when using prokaryotic cell-free expression systems. Eukaryotic Ribosome Display is potentially a more accessible alternative because of the availability of suitable commercial reagents, yet despite published protocols, this method has been less widely used. For eukaryotic Ribosome Display, a novel mechanism of mRNA recovery compared with that of the well-proven prokaryotic method has been proposed. We have examined the eukaryotic Ribosome Display process with the aims of investigating the proposed mechanism of sequence recovery and of identifying aspects of the protocol that may have lead to poor performance and therefore so far limited its use. We demonstrate that the proposed novel method is in fact mechanistically comparable to the prokaryotic method and we provide a step-by-step protocol for eukaryotic Ribosome Display that is 20-fold more efficient than current published methods. Our findings should increase the ease of operating Ribosome Display technology, making it more accessible to the scientific community.
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selection of hapten specific single domain antibodies from a non immunized llama Ribosome Display library
Journal of Immunological Methods, 2003Co-Authors: Maria Groves, Shenghua Li, Jamshid Tanha, Claudia Sheedy, Lutz Jermutus, Roger C Mackenzie, Christopher J HallAbstract:Picloram-specific variable fragments (V(HH)s) of heavy chain antibodies (HCAbs) were selected from a nai;ve-llama library using Ribosome Display technology. A cDNA library of V(HH)s was constructed from lymphocytes of a non-immunized llama and engineered to allow in vitro transcription and translation. With no stop codons present on the transcripts, trimeric complexes of Ribosomes, mRNAs and nascent peptides were produced for affinity selection, i.e. panning. After three cycles of panning, seven different V(HH)s all belonging to the V(HH) subfamily 1 were isolated. Following another three cycles of selection, only two of the seven V(HH)s persisted. A comparison of these two sequences with known sequences in the literature suggests that point mutations may have been introduced into the DNA pool during PCR amplification steps of library construction, panning and/or cloning. Three separate point mutations causing three independent amino acid changes (nonsynonomous mutations) accumulated in the same sequence and enriched throughout the selection protocol, suggesting that these changes confer binding advantages. Surface plasmon resonance (SPR) analysis was used to determine binding kinetics of the two clones (3-1D2 and 3-1F6) representing the two different sets of isolated complementarity determining region (CDR)3s. Measured K(D)s were 3 and 254 muM, respectively. The results indicate that Ribosome Display technology can be used to efficiently isolate hapten-specific antibody (Ab) fragments from a nai;ve library and concurrently introduce diversity to the selected pool thereby facilitating molecular evolution. Ribosome Display technology can compensate for the limited diversity of a V(HH) nai;ve library and provide an unlimited source of affinity-matured immunoactive reagents in vitro.
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Selection of hapten-specific single-domain antibodies from a non-immunized llama Ribosome Display library
Journal of Immunological Methods, 2003Co-Authors: Kerrm Y.f. Yau, Maria A.t. Groves, Shenghua Li, Hung Lee, Jamshid Tanha, Colin R Mackenzie, Claudia Sheedy, Lutz Jermutus, J. Christopher HallAbstract:Picloram-specific variable fragments (VHHs) of heavy chain antibodies (HCAbs) were selected from a naïve-llama library using Ribosome Display technology. A cDNA library of VHHs was constructed from lymphocytes of a non-immunized llama and engineered to allow in vitro transcription and translation. With no stop codons present on the transcripts, trimeric complexes of Ribosomes, mRNAs and nascent peptides were produced for affinity selection, i.e. panning. After three cycles of panning, seven different VHHs all belonging to the VHH subfamily 1 were isolated. Following another three cycles of selection, only two of the seven VHHs persisted. A comparison of these two sequences with known sequences in the literature suggests that point mutations may have been introduced into the DNA pool during PCR amplification steps of library construction, panning and/or cloning. Three separate point mutations causing three independent amino acid changes (nonsynonomous mutations) accumulated in the same sequence and enriched throughout the selection protocol, suggesting that these changes confer binding advantages. Surface plasmon resonance (SPR) analysis was used to determine binding kinetics of the two clones (3-1D2 and 3-1F6) representing the two different sets of isolated complementarity determining region (CDR)3s. Measured KDs were 3 and 254 μM, respectively. The results indicate that Ribosome Display technology can be used to efficiently isolate hapten-specific antibody (Ab) fragments from a naïve library and concurrently introduce diversity to the selected pool thereby facilitating molecular evolution. Ribosome Display technology can compensate for the limited diversity of a VHH naïve library and provide an unlimited source of affinity-matured immunoactive reagents in vitro. © 2003 Elsevier B.V. All rights reserved.
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in vitro selection for catalytic activity with Ribosome Display
Journal of the American Chemical Society, 2002Co-Authors: Patrick Amstutz, Lutz Jermutus, Joelle N Pelletier, Armin Guggisberg, Sandro Cesarotadic, Christian Zahnd, Andreas PluckthunAbstract:We report what is, to our knowledge, the first in vitro selection for catalytic activity based on catalytic turnover by using Ribosome Display, a method which does not involve living cells at any s...
