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

DOI: 10.1016/j.bbrc.2008.04.182 DOI Help
PMID: 18474222 PMID Help

Authors: Keisuke Ito (RIKEN SPring-8 Center) , Taishi Sugawara (University of Tokyo) , Mitsumorni Shiroishi (Japan Science and Technology Agency) , Natsuko Tokuda (Kyoto University) , Azusa Kurokawa (The University of Tokyo) , Takeshi Misaka (University of Tokyo) , Hisayoshi Makyio (Japan Science and Technology Agency) , Takami Yurugi-kobayashi (Japan Science and Technology Agency) , Tatsuro Shimamura (Japan Science and Technology Agency) , Norimichi Nomura (Japan Science and Technology Agency) , Takeshi Murata , Keiko Abe , So Iwata (Japan Science and Technology Agency; Kyoto University; RIKEN Genomic Sciences Center; Imperial College, London;) , Takuya Kobayashi (Japan Science and Technology Agency; Kyoto University)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Biochemical And Biophysical Research Communications , VOL 371 (4) , PAGES 841-5.

State: Published (Approved)
Published: July 2008

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.

Journal Keywords: High-Throughput; Protein Expression; Site-Directed Mutagenesis; Eukaryotic Membrane Protein; Crystallization; Saccharomyces Cerevisiae; Gfp-Fusion; G Protein-Coupled Receptor; Taste

Subject Areas: Biology and Bio-materials

Instruments: NONE-No attached Diamond beamline

Added On: 19/08/2009 23:07

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