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: ジャーナルへの寄稿記事

26 引用 (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.

元の言語英語
ページ(範囲)841-845
ページ数5
ジャーナルBiochemical and Biophysical Research Communications
371
発行部数4
DOI
出版物ステータス出版済み - 7 11 2008

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Yeast
Saccharomyces cerevisiae
Membrane Proteins
Yeasts
Homologous Recombination
Throughput
Crystallization
Polymerase Chain Reaction
Fusion reactions
Mutation
Cloning
Mutant Proteins
Genetic Recombination
Organism Cloning
Plasmids
Clone Cells
Genes
Diffraction
Proteins

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

これを引用

Advanced method for high-throughput expression of mutated eukaryotic membrane proteins in Saccharomyces cerevisiae. / Ito, Keisuke; Sugawara, Taishi; Shiroishi, Mitsunori; Tokuda, Natsuko; Kurokawa, Azusa; Misaka, Takumi; Makyio, Hisayoshi; Yurugi-Kobayashi, Takami; Shimamura, Tatsuro; Nomura, Norimichi; Murata, Takeshi; Abe, Keiko; Iwata, So; Kobayashi, Takuya.

:: Biochemical and Biophysical Research Communications, 巻 371, 番号 4, 11.07.2008, p. 841-845.

研究成果: ジャーナルへの寄稿記事

Ito, K, Sugawara, T, Shiroishi, M, Tokuda, N, Kurokawa, A, Misaka, T, Makyio, H, Yurugi-Kobayashi, T, Shimamura, T, Nomura, N, Murata, T, Abe, K, Iwata, S & Kobayashi, T 2008, 'Advanced method for high-throughput expression of mutated eukaryotic membrane proteins in Saccharomyces cerevisiae', Biochemical and Biophysical Research Communications, 巻. 371, 番号 4, pp. 841-845. https://doi.org/10.1016/j.bbrc.2008.04.182
Ito, Keisuke ; Sugawara, Taishi ; Shiroishi, Mitsunori ; Tokuda, Natsuko ; Kurokawa, Azusa ; Misaka, Takumi ; Makyio, Hisayoshi ; Yurugi-Kobayashi, Takami ; Shimamura, Tatsuro ; Nomura, Norimichi ; Murata, Takeshi ; Abe, Keiko ; Iwata, So ; Kobayashi, Takuya. / Advanced method for high-throughput expression of mutated eukaryotic membrane proteins in Saccharomyces cerevisiae. :: Biochemical and Biophysical Research Communications. 2008 ; 巻 371, 番号 4. pp. 841-845.
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AU - Kurokawa, Azusa

AU - Misaka, Takumi

AU - Makyio, Hisayoshi

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AU - Shimamura, Tatsuro

AU - Nomura, Norimichi

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AU - Abe, Keiko

AU - Iwata, So

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