ArticleBiotechnology for biofuels2016
Development of simple random mutagenesis protocol for the protein expression system in Pichia pastoris.
Article in Biotechnology for biofuels, 2016. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 18 papers.
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18 citing papers in PubMed, 36 citations in OpenAlex.
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- Recombinant production of SAG1 fused with xylanase in Pichia pastoris induced higher protective immunity against Eimeria tenella infection in chicken.Microbial biotechnology · 2024Article
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- Thermotolerance Mechanism of Fungal GH6 Cellobiohydrolase. Part I. Characterization of Thermotolerant Mutant from the BasidiomyceteJournal of applied glycoscience · 2024Article
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- Fermentation, Purification, and Tumor Inhibition of a Disulfide-Stabilized Diabody Against Fibroblast Growth Factor-2.Frontiers in oncology · 2021Article
- Pichia pastoris: A highly successful expression system for optimal synthesis of heterologous proteins.Journal of cellular physiology · 2020Review
- Thermostable Mutants of Glycoside Hydrolase Family 6 Cellobiohydrolase from the BasidiomyceteJournal of applied glycoscience · 2020Article
- Single Amino Acid Mutation of Pyranose 2-Oxidase Results in Increased Specificity for Diabetes Biomarker 1,5-Anhydro-D-Glucitol.Journal of applied glycoscience · 2020Article
- Crystal structure of a family 6 cellobiohydrolase from the basidiomycete Phanerochaete chrysosporium.Acta crystallographica. Section F, Structural biology communications · 2017Article
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Authors and funding
8 authors at 2 institutions in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
backgroundRandom 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.
resultsWe 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.
conclusionsWe 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.
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