Nakayama Manabu, Ohara Osamu
Department of Human Gene Research, Kazusa DNA Research Institute, Chiba, Japan.
Biochem Biophys Res Commun. 2003 Dec 19;312(3):825-30. doi: 10.1016/j.bbrc.2003.10.193.
Protein insolubility is a major problem when producing recombinant proteins (e.g., to be used as antigens) from large cDNAs in Escherichia coli. Here, we describe a system using three convertible plasmid vectors to screen for soluble proteins produced in E. coli. This system experimentally identified any random cDNA fragments producing soluble protein domains. Shotgun fragments introduced into any of our three plasmids, which contain Gateway recombination sites, fused in-frame to the ORF of the protein tag. These plasmids produced N-terminal GST- and C-terminal three-frame-adaptive FLAG-tagged proteins, kanamycin-resistant gene-tagged proteins (which were pre-selected for in-frame fused cDNAs), or GFP-tagged fusion proteins. The latter is useful as a fluorescence indicator of protein folding. The Gateway recombination sites promote smooth conversion for enrichment of in-frame clones and facilitate both protein solubility assays and final production of proteins without the C-terminal tag. This high-throughput screening method is particularly useful for procedures that require the handling of many cDNAs in parallel.
在大肠杆菌中从大的互补DNA（例如用作抗原）生产重组蛋白时，蛋白质不溶性是一个主要问题。在此，我们描述了一种使用三种可转换质粒载体来筛选大肠杆菌中产生的可溶性蛋白的系统。该系统通过实验鉴定出任何产生可溶性蛋白结构域的随机cDNA片段。引入我们三个质粒中任何一个的鸟枪法片段，这些质粒含有Gateway重组位点，与蛋白标签的开放阅读框（ORF）进行读码框内融合。这些质粒产生N端带有谷胱甘肽S-转移酶（GST）和C端带有三框适应性FLAG标签的蛋白、带有卡那霉素抗性基因标签的蛋白（预先筛选用于读码框内融合的cDNA）或GFP标签的融合蛋白。后者用作蛋白质折叠的荧光指示剂。Gateway重组位点促进读码框内克隆富集的顺利转换，并便于进行蛋白质溶解度测定以及最终生产不含C端标签的蛋白质。这种高通量筛选方法对于需要并行处理许多cDNA的程序特别有用。
