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Development of simple random mutagenesis protocol for the protein expression system in Pichia pastoris

Overview of attention for article published in Biotechnology for Biofuels and Bioproducts, September 2016
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Title
Development of simple random mutagenesis protocol for the protein expression system in Pichia pastoris
Published in
Biotechnology for Biofuels and Bioproducts, September 2016
DOI 10.1186/s13068-016-0613-z
Pubmed ID
Authors

Mikako Tachioka, Naohisa Sugimoto, Akihiko Nakamura, Naoki Sunagawa, Takuya Ishida, Taku Uchiyama, Kiyohiko Igarashi, Masahiro Samejima

Abstract

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. 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. 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.

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Mendeley readers

Mendeley readers

The data shown below were compiled from readership statistics for 126 Mendeley readers of this research output. Click here to see the associated Mendeley record.

Geographical breakdown

Country Count As %
Japan 1 <1%
Austria 1 <1%
Unknown 124 98%

Demographic breakdown

Readers by professional status Count As %
Student > Bachelor 26 21%
Researcher 17 13%
Student > Ph. D. Student 16 13%
Student > Master 12 10%
Student > Doctoral Student 5 4%
Other 11 9%
Unknown 39 31%
Readers by discipline Count As %
Biochemistry, Genetics and Molecular Biology 40 32%
Agricultural and Biological Sciences 22 17%
Unspecified 4 3%
Immunology and Microbiology 3 2%
Medicine and Dentistry 3 2%
Other 13 10%
Unknown 41 33%
Attention Score in Context

Attention Score in Context

This research output has an Altmetric Attention Score of 2. This is our high-level measure of the quality and quantity of online attention that it has received. This Attention Score, as well as the ranking and number of research outputs shown below, was calculated when the research output was last mentioned on 16 December 2016.
All research outputs
#15,740,207
of 25,374,647 outputs
Outputs from Biotechnology for Biofuels and Bioproducts
#862
of 1,578 outputs
Outputs of similar age
#188,301
of 328,307 outputs
Outputs of similar age from Biotechnology for Biofuels and Bioproducts
#18
of 37 outputs
Altmetric has tracked 25,374,647 research outputs across all sources so far. This one is in the 37th percentile – i.e., 37% of other outputs scored the same or lower than it.
So far Altmetric has tracked 1,578 research outputs from this source. They receive a mean Attention Score of 4.9. This one is in the 44th percentile – i.e., 44% of its peers scored the same or lower than it.
Older research outputs will score higher simply because they've had more time to accumulate mentions. To account for age we can compare this Altmetric Attention Score to the 328,307 tracked outputs that were published within six weeks on either side of this one in any source. This one is in the 41st percentile – i.e., 41% of its contemporaries scored the same or lower than it.
We're also able to compare this research output to 37 others from the same source and published within six weeks on either side of this one. This one has gotten more attention than average, scoring higher than 51% of its contemporaries.