Advanced method for high-throughput expression of mutated eukaryotic membrane proteins in Saccharomyces cerevisiae

Keisuke Ito, Taishi Sugawara, Mitsunori Shiroishi, Natsuko Tokuda, Azusa Kurokawa, Takumi Misaka, Hisayoshi Makyio, Takami Yurugi-Kobayashi, Tatsuro Shimamura, Norimichi Nomura, Takeshi Murata, Keiko Abe, So Iwata, Takuya Kobayashi

研究成果: Contribution to journalArticle査読

27 被引用数 (Scopus)

抄録

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.

本文言語英語
ページ(範囲)841-845
ページ数5
ジャーナルBiochemical and Biophysical Research Communications
371
4
DOI
出版ステータス出版済み - 7 11 2008

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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