title: High-throughput plasmid construction using homologous recombination in yeast: its mechanisms and application to protein production for X-ray crystallography.
abstract: Homologous recombination is a system for repairing the broken genomes of living organisms by connecting two DNA strands at their homologous sequences. Today, homologous recombination in yeast is used for plasmid construction as a substitute for traditional methods using restriction enzymes and ligases. This method has various advantages over the traditional method, including flexibility in the position of DNA insertion and ease of manipulation. Recently, the author of this review reported the construction of plasmids by homologous recombination in the methanol-utilizing yeast Pichia pastoris, which is known to be an excellent expression host for secretory proteins and membrane proteins. The method enabled high-throughput construction of expression systems of proteins using P. pastoris; the constructed expression systems were used to investigate the expression conditions of membrane proteins and to perform X-ray crystallography of secretory proteins. This review discusses the mechanisms and applications of homologous recombination, including the production of proteins for X-ray crystallography.
authors.affiliation: a Laboratory of Applied Structural Biology, Division of Applied Life Sciences, Graduate School of Agriculture , Kyoto University , Kyoto , Japan.
authors.forename: Kimihiko
authors.initials: K
authors.lastname: Mizutani
mesh_terms: D018360:Crystallography, X-Ray; D015870:Gene Expression; D005822:Genetic Vectors / Q000737:chemistry* / Q000378:metabolism; D059765:Homologous Recombination; D008565:Membrane Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D000432:Methanol / Q000378:metabolism; D008958:Models, Molecular; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D019076:Transgenes
chemical_list: D008565:Membrane Proteins; D011994:Recombinant Proteins; D000432:Methanol
keywords: Pichia pastoris; X-ray crystallography; expression system; homologous recombination; plasmid construction