Advanced method for high-throughput expression of mutated eukaryotic membrane proteins in Saccharomyces cerevisiae
- 作者：Keisuke Ito ; Taishi Sugawara ; Mitsunori Shiroishi ; Natsuko Tokuda ; Azusa Kurokawa ; Takumi Misaka ; Hisayoshi Makyio ; Takami Yurugi-Kobayashi ; Tatsuro Shimamura ; Norimichi Nomura ; Takeshi Murata ; Keiko
- 关键词：High-throughput ; Protein expression ; Site-directed mutagenesis ; Eukaryotic membrane protein ; Crystallization ; Saccharomyces cerevisiae ; GFP-fusion ; G protein-coupled receptor ; Taste
- 刊名：Biochemical and Biophysical Research Communications
- 出版年：2008
- 期刊代码：159_0006291x
- 类别：bio
- 出版时间：11 July 2008
- 卷：371
- 期：4
- 页码：841-845
- 文件大小：426 K
摘要
Crystallization of eukaryotic membrane proteins is a challenging, iterative process. The protein of interest is often modified in an attempt to improve crystallization and diffraction results. To accelerate this process, we took advantage of a GFP-fusion yeast expression system that uses PCR to direct homologous recombination and gene cloning. We explored the possibility of employing more than one PCR fragment to introduce various mutations in a single step, and found that when up to five PCR fragments were co-transformed into yeast, the recombination frequency was maintained as the number of fragments was increased. All transformants expressed the model membrane protein, while the resulting plasmid from each clone contained the designed mutations only. Thus, we have demonstrated a technique allowing the expression of mutant membrane proteins within 5 days, combining a GFP-fusion expression system and yeast homologous recombination.