The PRESAT-vector: Asymmetric T-vector for high-throughput screening of soluble protein domains for structural proteomics
The PRESAT-vector: Asymmetric T-vector for high-throughput screening of soluble protein domains for structural proteomics
复制标题
DOI：
10.1110/ps.03439004
复制
发表时间：
2004-03-01
期刊：
影响因子：
8
通讯作者：
Shirakawa, M
中科院分区：
文献类型：
--
作者：
Goda, N;Tenno, T;Shirakawa, M
描述了一种用于将PCR扩增的cDNA片段克隆到几乎任何融合蛋白表达载体中的快速单向方法。该方法，称为PRESAT载体克隆，是基于T载体技术，不需要限制性内切酶消化的PCR产物。随后，我们应用了一种新的ORF选择方法的连接质粒产物。第二步涉及限制性内切酶处理，其在转化到细菌宿主中用于进一步的蛋白质表达研究之前消除含有错误方向的ORF的质粒。为了实现这种选择，我们定制了对应于待表达蛋白质C末端的“后”PCR引物的5 '序列。集落以>90%的效率仅含有所需取向的连接产物。将该方法应用于GST融合表达系统，并对来自表达文库的可溶性蛋白的HTS系统进行了测试。
A rapid unidirectional method for cloning PCR-amplified cDNA fragments into virtually any fusion protein expression vector is described. The method, termed PRESAT-vector cloning, is based on a T-vector technique that does not require restriction endonuclease digestion of the PCR product. Subsequently, we applied a novel ORF selection method of the ligated plasmid products. This second step involves restriction endonuclease treatment that eliminates the plasmids containing an ORF in the wrong orientation prior to transformation into the bacterial host for further protein expression studies. To achieve this selection, we customized the 5'-sequence of the "rear" PCR primer corresponding to the C terminus of the protein to be expressed. The colonies harbored only the ligated products of the desired orientation at >90% efficiency. This method is applied to a GST fusion expression system, and an HTS system for soluble proteins from an expression library was tested.