論文 - 詳細
| RRC ID | 10772 |
|---|---|
| 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 |
| WOS 分野 | BIOTECHNOLOGY & APPLIED MICROBIOLOGY BIOCHEMICAL RESEARCH METHODS BIOCHEMISTRY & MOLECULAR BIOLOGY |
| オルトメトリクス指標 |
オルトメトリクス指標項目
|
| 各媒体での言及数の合計 | 0 |
| 過去6か月間でのオルトメトリクス指標の変動値 | 0.0 |
| リソース情報 | |
| メダカ | full length cDNA (olecno10_i02 olecno3_i19 olebno16_g13 olebrno16_g02 olebno62_l03) |