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Development of simple random mutagenesis protocol for the protein expression system in Pichia pastoris
BACKGROUND: Random mutagenesis is a powerful technique to obtain mutant proteins with different properties from the wild-type molecule. Error-prone PCR is often employed for random mutagenesis in bacterial protein expression systems, but has rarely been used in the methylotrophic yeast Pichia pastor...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2016
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5028916/ https://www.ncbi.nlm.nih.gov/pubmed/27660653 http://dx.doi.org/10.1186/s13068-016-0613-z |
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author | Tachioka, Mikako Sugimoto, Naohisa Nakamura, Akihiko Sunagawa, Naoki Ishida, Takuya Uchiyama, Taku Igarashi, Kiyohiko Samejima, Masahiro |
author_facet | Tachioka, Mikako Sugimoto, Naohisa Nakamura, Akihiko Sunagawa, Naoki Ishida, Takuya Uchiyama, Taku Igarashi, Kiyohiko Samejima, Masahiro |
author_sort | Tachioka, Mikako |
collection | PubMed |
description | BACKGROUND: Random mutagenesis is a powerful technique to obtain mutant proteins with different properties from the wild-type molecule. Error-prone PCR is often employed for random mutagenesis in bacterial protein expression systems, but has rarely been used in the methylotrophic yeast Pichia pastoris system, despite its significant advantages, mainly because large (μg-level) amounts of plasmids are required for transformation. RESULTS: We developed a quick and easy technique for random mutagenesis in P. pastoris by sequential Phi29 DNA polymerase-based amplification methods, error-prone rolling circle amplification (RCA) and multiple displacement amplification (MDA). The methodology was validated by applying it for random mutation of the gene encoding cellulase from the basidiomycete Phanerochaete chrysosporium (PcCel6A), a key enzyme in degradation of cellulosic biomass. In the error-prone RCA step, the concentrations of manganese ion (Mn(2+)) and cellulase gene-containing plasmid were varied, and the products obtained under each condition were subjected to the second MDA step in the absence of Mn(2+). The maximum error rate was 2.6 mutations/kb, as evaluated from the results of large-scale sequencing. Several μg of MDA products was transformed by electroporation into Pichia cells, and the activities of extracellularly expressed PcCel6A mutants towards crystalline and amorphous celluloses were compared with those of wild-type enzyme to identify key amino acid residues affecting degradation of crystalline cellulose. CONCLUSIONS: We present a rapid and convenient random mutagenesis method that does not require laborious steps such as ligation, cloning, and synthesis of specific primers. This method was successfully applied to the protein expression system in P. pastoris. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13068-016-0613-z) contains supplementary material, which is available to authorized users. |
format | Online Article Text |
id | pubmed-5028916 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-50289162016-09-22 Development of simple random mutagenesis protocol for the protein expression system in Pichia pastoris Tachioka, Mikako Sugimoto, Naohisa Nakamura, Akihiko Sunagawa, Naoki Ishida, Takuya Uchiyama, Taku Igarashi, Kiyohiko Samejima, Masahiro Biotechnol Biofuels Methodology BACKGROUND: Random mutagenesis is a powerful technique to obtain mutant proteins with different properties from the wild-type molecule. Error-prone PCR is often employed for random mutagenesis in bacterial protein expression systems, but has rarely been used in the methylotrophic yeast Pichia pastoris system, despite its significant advantages, mainly because large (μg-level) amounts of plasmids are required for transformation. RESULTS: We developed a quick and easy technique for random mutagenesis in P. pastoris by sequential Phi29 DNA polymerase-based amplification methods, error-prone rolling circle amplification (RCA) and multiple displacement amplification (MDA). The methodology was validated by applying it for random mutation of the gene encoding cellulase from the basidiomycete Phanerochaete chrysosporium (PcCel6A), a key enzyme in degradation of cellulosic biomass. In the error-prone RCA step, the concentrations of manganese ion (Mn(2+)) and cellulase gene-containing plasmid were varied, and the products obtained under each condition were subjected to the second MDA step in the absence of Mn(2+). The maximum error rate was 2.6 mutations/kb, as evaluated from the results of large-scale sequencing. Several μg of MDA products was transformed by electroporation into Pichia cells, and the activities of extracellularly expressed PcCel6A mutants towards crystalline and amorphous celluloses were compared with those of wild-type enzyme to identify key amino acid residues affecting degradation of crystalline cellulose. CONCLUSIONS: We present a rapid and convenient random mutagenesis method that does not require laborious steps such as ligation, cloning, and synthesis of specific primers. This method was successfully applied to the protein expression system in P. pastoris. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13068-016-0613-z) contains supplementary material, which is available to authorized users. BioMed Central 2016-09-19 /pmc/articles/PMC5028916/ /pubmed/27660653 http://dx.doi.org/10.1186/s13068-016-0613-z Text en © The Author(s) 2016 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. |
spellingShingle | Methodology Tachioka, Mikako Sugimoto, Naohisa Nakamura, Akihiko Sunagawa, Naoki Ishida, Takuya Uchiyama, Taku Igarashi, Kiyohiko Samejima, Masahiro Development of simple random mutagenesis protocol for the protein expression system in Pichia pastoris |
title | Development of simple random mutagenesis protocol for the protein expression system in Pichia pastoris |
title_full | Development of simple random mutagenesis protocol for the protein expression system in Pichia pastoris |
title_fullStr | Development of simple random mutagenesis protocol for the protein expression system in Pichia pastoris |
title_full_unstemmed | Development of simple random mutagenesis protocol for the protein expression system in Pichia pastoris |
title_short | Development of simple random mutagenesis protocol for the protein expression system in Pichia pastoris |
title_sort | development of simple random mutagenesis protocol for the protein expression system in pichia pastoris |
topic | Methodology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5028916/ https://www.ncbi.nlm.nih.gov/pubmed/27660653 http://dx.doi.org/10.1186/s13068-016-0613-z |
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