RRC ID 10773
Author Mizutani K, Yoshioka S, Mizutani Y, Iwata S, Mikami B.
Title High-throughput construction of expression system using yeast Pichia pastoris, and its application to membrane proteins.
Journal 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).
Volume 77(1)
Pages 1-8
Published 2011-5
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 1.338
Times Cited 12
WOS Category BIOTECHNOLOGY & APPLIED MICROBIOLOGY BIOCHEMICAL RESEARCH METHODS BIOCHEMISTRY & MOLECULAR BIOLOGY
Resource
Yeast BY2778 BY21467