Bannasch D, Schwab M
Division of Cytogenetics-H0400, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, Heidelberg, D-69120, Germany.
Plasmid. 1999 Sep;42(2):139-43. doi: 10.1006/plas.1999.1413.
Two-hybrid screens have been widely used in recent years for identifying and isolating new interaction partners to known proteins. Current Gal4-dependent two-hybrid screens employ mostly bait and library vectors, which both have the ampicillin gene as a selection marker in bacteria. The isolation of the library plasmids takes several days, because library and bait plasmid cannot be separated easily. We have replaced the ampicillin gene by a kanamycin gene in a Gal4 DNA binding domain bait vector. This vector reduces four- to fivefold the time period required for the isolation of library plasmid DNA. In addition we have changed the multicloning site in the modified vector for easy cloning of cDNA inserts. This vector is advantageous not only in standard two-hybrid screens, but also in mass screens that require multiple screening rounds in order to characterize networks of protein-protein interactions.
近年来，双杂交筛选已被广泛用于识别和分离已知蛋白质的新相互作用伙伴。目前依赖Gal4的双杂交筛选主要使用诱饵和文库载体，二者在细菌中均以氨苄青霉素基因作为选择标记。文库质粒的分离需要数天时间，因为文库质粒和诱饵质粒不易分离。我们在Gal4 DNA结合结构域诱饵载体中用卡那霉素基因取代了氨苄青霉素基因。该载体将分离文库质粒DNA所需的时间缩短了四至五倍。此外，我们还改变了修饰载体中的多克隆位点，以便于cDNA插入片段的克隆。该载体不仅在标准双杂交筛选中具有优势，而且在需要多轮筛选以表征蛋白质-蛋白质相互作用网络的大规模筛选中也具有优势。
