a three-hybrid system to probe in vivo protein-protein interactions application to the essential proteins of the rd1 complex of m. tuberculosis三辆混合动力系统探测体内蛋白质交互应用程序的基本蛋白质rd1结核分枝杆菌的复杂.pdfVIP

A Three-Hybrid System to Probe In Vivo Protein-Protein Interactions: Application to the Essential Proteins of the RD1 Complex of M. tuberculosis . . Megha Tharad , Sachin Kumar Samuchiwal , Kuhulika Bhalla, Anamika Ghosh, Krishan Kumar, Sushil Kumar, Anand Ranganathan* Recombinant Gene Products Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, India Abstract Background: Protein-protein interactions play a crucial role in enabling a pathogen to survive within a host. In many cases the interactions involve a complex of proteins rather than just two given proteins. This is especially true for pathogens like M. tuberculosis that are able to successfully survive the inhospitable environment of the macrophage. Studying such interactions in detail may help in developing small molecules that either disrupt or augment the interactions. Here, we describe the development of an E. coli based bacterial three-hybrid system that can be used effectively to study ternary protein complexes. Methodology/Principal Findings: The protein-protein interactions involved in M. tuberculosis pathogenesis have been used as a model for the validation of the three-hybrid system. Using the M. tuberculosis RD1 encoded proteins CFP10, ESAT6 and Rv3871 for our proof-of-concept studies, we show that the interaction between the proteins CFP10 and Rv3871 is strengthened and stabilized in the presence of ESAT6, the known heterodimeric partner of CFP10. Isolating peptide candidates that can disrupt crucial protein-protein interactions is another application that the system offers. We demonstrate this by using CFP10 protein as a disruptor of a previously es