Advanced method for high-throughput expression of mutated eukaryotic membrane proteins in Saccharomyces cerevisiae
| Autor: | So Iwata, Takeshi Murata, Hisayoshi Makyio, Mitsunori Shiroishi, Tatsuro Shimamura, Azusa Kurokawa, Takuya Kobayashi, Natsuko Tokuda, Norimichi Nomura, Keiko Abe, Takumi Misaka, Takami Yurugi-Kobayashi, Taishi Sugawara, Keisuke Ito |
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| Rok vydání: | 2008 |
| Předmět: |
FLP-FRT recombination
Saccharomyces cerevisiae Green Fluorescent Proteins Biophysics Mutagenesis (molecular biology technique) TRPM Cation Channels Molecular cloning Biochemistry Polymerase Chain Reaction Mice Plasmid Transformation Genetic Animals Cloning Molecular Molecular Biology Recombination Genetic biology Membrane Proteins Cell Biology biology.organism_classification Molecular biology Yeast Recombinant Proteins Cell biology Membrane protein Protein Biosynthesis Mutation Homologous recombination |
| Zdroj: | Biochemical and biophysical research communications. 371(4) |
| ISSN: | 1090-2104 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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