Mizutani Kimihiko
a Laboratory of Applied Structural Biology, Division of Applied Life Sciences, Graduate School of Agriculture , Kyoto University , Kyoto , Japan.
Biosci Biotechnol Biochem. 2015;79(1):1-10. doi: 10.1080/09168451.2014.952614.
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.
同源重组是一种通过在两条DNA链的同源序列处连接来修复生物体断裂基因组的系统。如今，酵母中的同源重组被用于质粒构建，以替代使用限制性内切酶和连接酶的传统方法。该方法相对于传统方法具有多种优势，包括DNA插入位置的灵活性和操作简便性。最近，本综述的作者报道了在利用甲醇的毕赤酵母中通过同源重组构建质粒，毕赤酵母是已知的分泌蛋白和膜蛋白的优秀表达宿主。该方法能够高通量构建利用毕赤酵母的蛋白质表达系统；构建的表达系统用于研究膜蛋白的表达条件以及进行分泌蛋白的X射线晶体学分析。本综述讨论了同源重组的机制和应用，包括用于X射线晶体学分析的蛋白质生产。
