title: Advanced method for high-throughput expression of mutated eukaryotic membrane proteins in Saccharomyces cerevisiae.
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.
authors.affiliation: Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan.
authors.forename: Keisuke
authors.initials: K
authors.lastname: Ito
authors.forename: Taishi
authors.initials: T
authors.lastname: Sugawara
authors.forename: Mitsunori
authors.initials: M
authors.lastname: Shiroishi
authors.forename: Natsuko
authors.initials: N
authors.lastname: Tokuda
authors.forename: Azusa
authors.initials: A
authors.lastname: Kurokawa
authors.forename: Takumi
authors.initials: T
authors.lastname: Misaka
authors.forename: Hisayoshi
authors.initials: H
authors.lastname: Makyio
authors.forename: Takami
authors.initials: T
authors.lastname: Yurugi-Kobayashi
authors.forename: Tatsuro
authors.initials: T
authors.lastname: Shimamura
authors.forename: Norimichi
authors.initials: N
authors.lastname: Nomura
authors.forename: Takeshi
authors.initials: T
authors.lastname: Murata
authors.forename: Keiko
authors.initials: K
authors.lastname: Abe
authors.forename: So
authors.initials: S
authors.lastname: Iwata
authors.forename: Takuya
authors.initials: T
authors.lastname: Kobayashi
mesh_terms: D000818:Animals; D003001:Cloning, Molecular / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics; D051379:Mice; D009154:Mutation; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D050053:TRPM Cation Channels / Q000096:biosynthesis / Q000235:genetics; D014170:Transformation, Genetic
chemical_list: D008565:Membrane Proteins; D011994:Recombinant Proteins; D050053:TRPM Cation Channels; C498617:Trpm5 protein, mouse; D049452:Green Fluorescent Proteins