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Construction and Analysis of High-Complexity Ribosome Display Random Peptide Libraries
Random peptide libraries displayed on the ribosome are becoming a new tool for the in vitro selection of biologically relevant macromolecules, including epitopes, antagonists, enzymes, and cell-surface receptors. Ribosome display is a cell-free system of coupling individual nascent proteins (phenoty...
Autores principales: | , , , , , |
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Formato: | Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2008
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2373887/ https://www.ncbi.nlm.nih.gov/pubmed/18493302 http://dx.doi.org/10.1371/journal.pone.0002092 |
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author | Yang, Li-Min Wang, Jing-Lin Kang, Lin Gao, Shan Liu, Yan-hua Hu, Ting-Mao |
author_facet | Yang, Li-Min Wang, Jing-Lin Kang, Lin Gao, Shan Liu, Yan-hua Hu, Ting-Mao |
author_sort | Yang, Li-Min |
collection | PubMed |
description | Random peptide libraries displayed on the ribosome are becoming a new tool for the in vitro selection of biologically relevant macromolecules, including epitopes, antagonists, enzymes, and cell-surface receptors. Ribosome display is a cell-free system of coupling individual nascent proteins (phenotypes) to their corresponding mRNA (genotypes) by the formation of stable protein-ribosome-mRNA complexes and permitting the selection of a functional nascent protein by iterative cycles of panning and reverse transcription-polymerase chain reaction (RT-PCR) amplification in vitro. The complexity of the random peptide library is critical for the success of a panning experiment; greater the diversity of sequences within the library, the more likely it is that the library comprises sequences that can bind a given target with specific affinity. Here, we have used the cell-free system Escherichia coli S30 lysate to construct high-complexity random peptide libraries (>10(14) independent members) by introducing strategies that are different from the methods described by Mattheakis et al. and Lamla et al. The key step in our method is to produce nanomole (nmol) amounts of DNA elements that are necessary for in vitro transcription/translation by using PCR but not plasmid DNA. Library design strategies and protocols that facilitate rapid identification are also presented. |
format | Text |
id | pubmed-2373887 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2008 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-23738872008-05-21 Construction and Analysis of High-Complexity Ribosome Display Random Peptide Libraries Yang, Li-Min Wang, Jing-Lin Kang, Lin Gao, Shan Liu, Yan-hua Hu, Ting-Mao PLoS One Research Article Random peptide libraries displayed on the ribosome are becoming a new tool for the in vitro selection of biologically relevant macromolecules, including epitopes, antagonists, enzymes, and cell-surface receptors. Ribosome display is a cell-free system of coupling individual nascent proteins (phenotypes) to their corresponding mRNA (genotypes) by the formation of stable protein-ribosome-mRNA complexes and permitting the selection of a functional nascent protein by iterative cycles of panning and reverse transcription-polymerase chain reaction (RT-PCR) amplification in vitro. The complexity of the random peptide library is critical for the success of a panning experiment; greater the diversity of sequences within the library, the more likely it is that the library comprises sequences that can bind a given target with specific affinity. Here, we have used the cell-free system Escherichia coli S30 lysate to construct high-complexity random peptide libraries (>10(14) independent members) by introducing strategies that are different from the methods described by Mattheakis et al. and Lamla et al. The key step in our method is to produce nanomole (nmol) amounts of DNA elements that are necessary for in vitro transcription/translation by using PCR but not plasmid DNA. Library design strategies and protocols that facilitate rapid identification are also presented. Public Library of Science 2008-05-21 /pmc/articles/PMC2373887/ /pubmed/18493302 http://dx.doi.org/10.1371/journal.pone.0002092 Text en Yang et al. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. |
spellingShingle | Research Article Yang, Li-Min Wang, Jing-Lin Kang, Lin Gao, Shan Liu, Yan-hua Hu, Ting-Mao Construction and Analysis of High-Complexity Ribosome Display Random Peptide Libraries |
title | Construction and Analysis of High-Complexity Ribosome Display Random Peptide Libraries |
title_full | Construction and Analysis of High-Complexity Ribosome Display Random Peptide Libraries |
title_fullStr | Construction and Analysis of High-Complexity Ribosome Display Random Peptide Libraries |
title_full_unstemmed | Construction and Analysis of High-Complexity Ribosome Display Random Peptide Libraries |
title_short | Construction and Analysis of High-Complexity Ribosome Display Random Peptide Libraries |
title_sort | construction and analysis of high-complexity ribosome display random peptide libraries |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2373887/ https://www.ncbi.nlm.nih.gov/pubmed/18493302 http://dx.doi.org/10.1371/journal.pone.0002092 |
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