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In Vitro Selection of Fab Fragments by mRNA Display and Gene-Linking Emulsion PCR
In vitro selection by display methods has been an effective tool for engineering recombinant antibodies. mRNA display based on a cell-free translation system has the advantages of larger library sizes and quicker selection procedures compared with cell-based display methods such as phage display. Ho...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Hindawi Publishing Corporation
2012
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3461632/ https://www.ncbi.nlm.nih.gov/pubmed/23050123 http://dx.doi.org/10.1155/2012/371379 |
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author | Sumida, Takeshi Yanagawa, Hiroshi Doi, Nobuhide |
author_facet | Sumida, Takeshi Yanagawa, Hiroshi Doi, Nobuhide |
author_sort | Sumida, Takeshi |
collection | PubMed |
description | In vitro selection by display methods has been an effective tool for engineering recombinant antibodies. mRNA display based on a cell-free translation system has the advantages of larger library sizes and quicker selection procedures compared with cell-based display methods such as phage display. However, mRNA display has been limited to select single-chain polypeptides such as scFvs due to its characteristic of linking a nascent polypeptide with its encoding mRNA on the ribosome. Here we demonstrated a new way of selecting heterodimeric Fab fragments by using mRNA display combined with emulsion PCR. We designed a pair of complementary 5′ UTR sequences that can link the Fab heavy and light chain genes together by overlap-extension PCR in water-in-oil emulsions. We confirmed that two mRNA-displayed polypeptides for heavy and light chain of a model Fab fragment were associated into the active form and that a specific Fab fragment gene was enriched over 100-fold per round of a model affinity selection followed by the gene-linking emulsion PCR. We further performed directed evolution of Fab fragments with higher binding activity from a randomized Fab fragment library. |
format | Online Article Text |
id | pubmed-3461632 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2012 |
publisher | Hindawi Publishing Corporation |
record_format | MEDLINE/PubMed |
spelling | pubmed-34616322012-10-04 In Vitro Selection of Fab Fragments by mRNA Display and Gene-Linking Emulsion PCR Sumida, Takeshi Yanagawa, Hiroshi Doi, Nobuhide J Nucleic Acids Research Article In vitro selection by display methods has been an effective tool for engineering recombinant antibodies. mRNA display based on a cell-free translation system has the advantages of larger library sizes and quicker selection procedures compared with cell-based display methods such as phage display. However, mRNA display has been limited to select single-chain polypeptides such as scFvs due to its characteristic of linking a nascent polypeptide with its encoding mRNA on the ribosome. Here we demonstrated a new way of selecting heterodimeric Fab fragments by using mRNA display combined with emulsion PCR. We designed a pair of complementary 5′ UTR sequences that can link the Fab heavy and light chain genes together by overlap-extension PCR in water-in-oil emulsions. We confirmed that two mRNA-displayed polypeptides for heavy and light chain of a model Fab fragment were associated into the active form and that a specific Fab fragment gene was enriched over 100-fold per round of a model affinity selection followed by the gene-linking emulsion PCR. We further performed directed evolution of Fab fragments with higher binding activity from a randomized Fab fragment library. Hindawi Publishing Corporation 2012 2012-09-23 /pmc/articles/PMC3461632/ /pubmed/23050123 http://dx.doi.org/10.1155/2012/371379 Text en Copyright © 2012 Takeshi Sumida et al. https://creativecommons.org/licenses/by/3.0/ This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Research Article Sumida, Takeshi Yanagawa, Hiroshi Doi, Nobuhide In Vitro Selection of Fab Fragments by mRNA Display and Gene-Linking Emulsion PCR |
title |
In Vitro Selection of Fab Fragments by mRNA Display and Gene-Linking Emulsion PCR |
title_full |
In Vitro Selection of Fab Fragments by mRNA Display and Gene-Linking Emulsion PCR |
title_fullStr |
In Vitro Selection of Fab Fragments by mRNA Display and Gene-Linking Emulsion PCR |
title_full_unstemmed |
In Vitro Selection of Fab Fragments by mRNA Display and Gene-Linking Emulsion PCR |
title_short |
In Vitro Selection of Fab Fragments by mRNA Display and Gene-Linking Emulsion PCR |
title_sort | in vitro selection of fab fragments by mrna display and gene-linking emulsion pcr |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3461632/ https://www.ncbi.nlm.nih.gov/pubmed/23050123 http://dx.doi.org/10.1155/2012/371379 |
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