RRC ID |
10773
|
著者 |
Mizutani K, Yoshioka S, Mizutani Y, Iwata S, Mikami B.
|
タイトル |
High-throughput construction of expression system using yeast Pichia pastoris, and its application to membrane proteins.
|
ジャーナル |
Protein Expr Purif
|
Abstract |
The well-established method for high-throughput construction of an expression system of the yeast Saccharomyces cerevisiae uses homologous recombination between an expression plasmid and a target gene (with homologous regions of the plasmid on both ends added by PCR). This method has been widely used for membrane proteins using plasmids containing GFP, and has been successfully used to investigate the cellular localization and solubilization conditions of the proteins. Although the methanol-utilizing yeast Pichia pastoris is known as an excellent expression host, a method for high-throughput construction of an expression system like that in S. cerevisiae has not been reported. In this study, we have attempted to construct expression systems via homologous recombination in P. pastoris. The insertion of genes into a plasmid could be easily checked by colony-PCR. Expression systems for seven membrane proteins of medaka fish (Oryzias latipes) and yeast (S. cerevisiae) were constructed, and the expression of proteins was analyzed by fluorescence spectra, fluorescence microscopy, and SDS-PAGE (in-gel fluorescence detection).
|
巻・号 |
77(1)
|
ページ |
1-8
|
公開日 |
2011-5-1
|
DOI |
10.1016/j.pep.2010.12.009
|
PII |
S1046-5928(10)00347-5
|
PMID |
21172439
|
MeSH |
Cloning, Molecular / methods*
Electrophoresis, Polyacrylamide Gel
Green Fluorescent Proteins / biosynthesis
Green Fluorescent Proteins / chemistry
Green Fluorescent Proteins / genetics
High-Throughput Screening Assays / methods*
Membrane Proteins / biosynthesis*
Membrane Proteins / chemistry
Membrane Proteins / genetics
Pichia / genetics*
Pichia / metabolism*
Plasmids / genetics
Polymerase Chain Reaction
Recombinant Fusion Proteins / biosynthesis
Recombinant Fusion Proteins / chemistry
Recombinant Fusion Proteins / genetics
Spectrometry, Fluorescence
|
IF |
1.513
|
引用数 |
16
|
WOS 分野
|
BIOTECHNOLOGY & APPLIED MICROBIOLOGY
BIOCHEMICAL RESEARCH METHODS
BIOCHEMISTRY & MOLECULAR BIOLOGY
|
リソース情報 |
酵母 |
BY2778
BY21467 |