title: High-throughput construction of expression system using yeast Pichia pastoris, and its application to membrane proteins.
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).
authors.affiliation: Laboratory of Applied Structural Biology, Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan. kmizutani@kais.kyoto-u.ac.jp
authors.forename: Kimihiko
authors.initials: K
authors.lastname: Mizutani
authors.forename: Soshi
authors.initials: S
authors.lastname: Yoshioka
authors.forename: Yukiko
authors.initials: Y
authors.lastname: Mizutani
authors.forename: So
authors.initials: S
authors.lastname: Iwata
authors.forename: Bunzo
authors.initials: B
authors.lastname: Mikami
mesh_terms: D003001:Cloning, Molecular / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D057166:High-Throughput Screening Assays / Q000379:methods*; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D010843:Pichia / Q000235:genetics* / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D013050:Spectrometry, Fluorescence
chemical_list: D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins