Park Jong-Hwa, Back Jung Ho, Hahm Soo Hyun, Shim Hye-Young, Park Min Ju, Ko Sung Il, Han Ye Sun
Department of Advanced Technology Fusion and Bio/Molecular Informatics Center, Konkuk University, Seoul, Korea.
J Microbiol Biotechnol. 2007 Oct;17(10):1607-15.
We investigated the applicability of the TEM-1 beta- lactamase fragment complementation (BFC) system to develop a strategy for the screening of protein-protein interactions in bacteria. A BFC system containing a human Fas-associated death domain (hFADD) and human Fas death domain (hFasDD) was generated. The hFADD-hFasDD interaction was verified by cell survivability in ampicillin-containing medium and the colorimetric change of nitrocefin. It was also confirmed by His pull-down assay using cell lysates obtained in selection steps. A coiled-coil helix coiled-coil domain-containing protein 5 (CHCH5) was identified as an interacting protein of human uracil DNA glycosylase (hUNG) from the bacterial BFC cDNA library strategy. The interaction between hUNG and CHCH5 was further confirmed with immunoprecipitation using a mammalian expression system. CHCH5 enhanced the DNA glycosylase activity of hUNG to remove uracil from DNA duplexes containing a U/G mismatch pair. These results suggest that the bacterial BFC cDNA library strategy can be effectively used to identify interacting protein pairs.
我们研究了TEM-1β-内酰胺酶片段互补（BFC）系统在开发细菌中蛋白质-蛋白质相互作用筛选策略方面的适用性。构建了一个包含人Fas相关死亡结构域（hFADD）和人Fas死亡结构域（hFasDD）的BFC系统。通过在含氨苄青霉素的培养基中的细胞存活率以及头孢硝噻吩的比色变化验证了hFADD-hFasDD相互作用。在筛选步骤中获得的细胞裂解物进行的His下拉试验也证实了这一点。通过细菌BFC cDNA文库策略，鉴定出一种含卷曲螺旋螺旋结构域蛋白5（CHCH5）是人尿嘧啶DNA糖基化酶（hUNG）的相互作用蛋白。使用哺乳动物表达系统进行免疫沉淀进一步证实了hUNG与CHCH5之间的相互作用。CHCH5增强了hUNG从含有U/G错配的DNA双链体中去除尿嘧啶的DNA糖基化酶活性。这些结果表明，细菌BFC cDNA文库策略可有效地用于鉴定相互作用的蛋白对。
