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Plasmid-Based One-Pot Saturation Mutagenesis and Robot-Based Automated Screening for Protein Engineering
[Image: see text] We evaluated a method for protein engineering using plasmid-based one-pot saturation mutagenesis and robot-based automated screening. When the biases in nucleotides and amino acids were assessed for a loss-of-function point mutation in green fluorescent protein, the ratios of gain-...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Chemical Society
2018
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6130897/ https://www.ncbi.nlm.nih.gov/pubmed/30221239 http://dx.doi.org/10.1021/acsomega.8b00663 |
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author | Kawai, Fumihiro Nakamura, Akihiko Visootsat, Akasit Iino, Ryota |
author_facet | Kawai, Fumihiro Nakamura, Akihiko Visootsat, Akasit Iino, Ryota |
author_sort | Kawai, Fumihiro |
collection | PubMed |
description | [Image: see text] We evaluated a method for protein engineering using plasmid-based one-pot saturation mutagenesis and robot-based automated screening. When the biases in nucleotides and amino acids were assessed for a loss-of-function point mutation in green fluorescent protein, the ratios of gain-of-function mutants were not significantly different from the expected values for the primers among the three different suppliers. However, deep sequencing analysis revealed that the ratios of nucleotides in the primers were highly biased among the suppliers. Biases for NNB were less severe than for NNN. We applied this method to screen a fusion protein of two chitinases, ChiA and ChiB (ChiAB). Three NNB codons as well as tyrosine and serine (X(1)YSX(2)X(3)) were inserted to modify the surface structure of ChiAB. We observed significant amino acid bias at the X(3) position in water-soluble, active ChiAB-X(1)YSX(2)X(3) mutants. Examination of the crystal structure of one active mutant, ChiAB-FYSFV, revealed that the X(3) residue plays an important role in structure stabilization. |
format | Online Article Text |
id | pubmed-6130897 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2018 |
publisher | American Chemical Society |
record_format | MEDLINE/PubMed |
spelling | pubmed-61308972018-09-12 Plasmid-Based One-Pot Saturation Mutagenesis and Robot-Based Automated Screening for Protein Engineering Kawai, Fumihiro Nakamura, Akihiko Visootsat, Akasit Iino, Ryota ACS Omega [Image: see text] We evaluated a method for protein engineering using plasmid-based one-pot saturation mutagenesis and robot-based automated screening. When the biases in nucleotides and amino acids were assessed for a loss-of-function point mutation in green fluorescent protein, the ratios of gain-of-function mutants were not significantly different from the expected values for the primers among the three different suppliers. However, deep sequencing analysis revealed that the ratios of nucleotides in the primers were highly biased among the suppliers. Biases for NNB were less severe than for NNN. We applied this method to screen a fusion protein of two chitinases, ChiA and ChiB (ChiAB). Three NNB codons as well as tyrosine and serine (X(1)YSX(2)X(3)) were inserted to modify the surface structure of ChiAB. We observed significant amino acid bias at the X(3) position in water-soluble, active ChiAB-X(1)YSX(2)X(3) mutants. Examination of the crystal structure of one active mutant, ChiAB-FYSFV, revealed that the X(3) residue plays an important role in structure stabilization. American Chemical Society 2018-07-11 /pmc/articles/PMC6130897/ /pubmed/30221239 http://dx.doi.org/10.1021/acsomega.8b00663 Text en Copyright © 2018 American Chemical Society This is an open access article published under an ACS AuthorChoice License (http://pubs.acs.org/page/policy/authorchoice_termsofuse.html) , which permits copying and redistribution of the article or any adaptations for non-commercial purposes. |
spellingShingle | Kawai, Fumihiro Nakamura, Akihiko Visootsat, Akasit Iino, Ryota Plasmid-Based One-Pot Saturation Mutagenesis and Robot-Based Automated Screening for Protein Engineering |
title | Plasmid-Based One-Pot Saturation Mutagenesis and Robot-Based
Automated Screening for Protein Engineering |
title_full | Plasmid-Based One-Pot Saturation Mutagenesis and Robot-Based
Automated Screening for Protein Engineering |
title_fullStr | Plasmid-Based One-Pot Saturation Mutagenesis and Robot-Based
Automated Screening for Protein Engineering |
title_full_unstemmed | Plasmid-Based One-Pot Saturation Mutagenesis and Robot-Based
Automated Screening for Protein Engineering |
title_short | Plasmid-Based One-Pot Saturation Mutagenesis and Robot-Based
Automated Screening for Protein Engineering |
title_sort | plasmid-based one-pot saturation mutagenesis and robot-based
automated screening for protein engineering |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6130897/ https://www.ncbi.nlm.nih.gov/pubmed/30221239 http://dx.doi.org/10.1021/acsomega.8b00663 |
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