High-throughput plasmid construction using homologous recombination in yeast: its mechanisms and application to protein production for X-ray crystallography

Abstract

To cite this article: Kimihiko Mizutani (2015) High-throughput plasmid construction using homologous recombination in yeast: its mechanisms and application to protein production for X-ray crystallography, Bioscience, Biotechnology, and Biochemistry Award ReviewHigh-throughput plasmid construction using homologous recombination in yeast: its mechanisms and application to protein production for X-ray crystallography 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.Key words: homologous recombination; plasmid construction; Pichia pastoris; expression system; X-ray crystallographyTo repair their broken genomes, living organisms have a system connecting two DNA strands at their similar (homologous) sequences.1-3) This system is called homologous recombination, and it is employed for genetic recombination during meiosis. In recent years, homologous recombination in yeast has been used for plasmid construction (such as insertion of a target gene into a plasmid) and gene isolation (such as transformation-associated recombination cloning to directly capture genomic loci from Saccharomyces cerevisiae).4,5) Unlike with traditional methods using restriction enzymes and ligases, the method for plasmid construction using homologous recombination can insert a DNA fragment into an appropriate and favorable position of a plasmid. The regions of homology used for recombination need not be at the ends of the linearized plasmid.4) For example, using a plasmid with a green fluorescence protein (GFP) in the region downstream of the promoter, homologous recombination has been applied to high-throughput construction of a yeast S. cerevisiae expression system of GFP membrane protein fusions.6,7) This system has been efficiently used to investigate the expression, purification, and crystallization conditions of membrane proteins. As another example, a method was developed for converting a common Escherichia coli vector to an E. coli yeast shuttle vector to enable the construction of plasmid by ho...

PFN2, a novel marker of unfavorable prognosis, is a potential therapeutic target involved in esophageal squamous cell carcinoma

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Loss-of-function of an Arabidopsis NADPH pyrophosphohydrolase, AtNUDX19, impacts on the pyridine nucleotides status and confers photooxidative stress tolerance

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Transformation-associated recombination (TAR) cloning for genomics studies and synthetic biology

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