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

Research output: Contribution to journalArticle

25 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)841-845
Number of pages5
JournalBiochemical and Biophysical Research Communications
Volume371
Issue number4
DOIs
Publication statusPublished - Jul 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

Cite this

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.

In: Biochemical and Biophysical Research Communications, Vol. 371, No. 4, 11.07.2008, p. 841-845.

Research output: Contribution to journalArticle

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, vol. 371, no. 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. In: Biochemical and Biophysical Research Communications. 2008 ; Vol. 371, No. 4. pp. 841-845.
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