Detecting of some YopM-protein interactions using fluorescence complementation assay

There are only three human pathogenic species of the genus Yersinia: Y.pestis, Y. enterocolitica and Y. pseudotuberculosis. These strains inhibit some of the host defense mechanisms, such as phagocytosis. Pathogenic strains of Yersinia has the ability to escape from the immune defense is by secreting effector proteins. YopM including leucine-rich repeating motifs is one of these effector proteins, and it is an acidic protein. It has been shown that this protein is able to penetrate the cell barrier, localize in nucleus and transport the protein. In this thesis study, protein-protein interaction between YopM and Yersinia spp. Type III secretion regulator proteins LcrV and LcrG was investigated based on the complementation of green fluorescent protein (GFP). Fusion PCR was used to fuse genes coding for N-terminal GFP fragment (1-157.amino acids) and LcrV/LcrG proteins. Similarly, the fusion gene fragment of C-terminal GFP (158-238. amino acids) and YopM was obtained. The two plasmids coding for NGFP-LcrG (or NGFP-LcrV) and YopM-CGFP were co-transformed into E. coli cells and the fluorecsence was monitored. Moreover, the fusion protein was purified and GFP fluorescence spectrum was monitored. As a result of interaction between YopM and LcrG, complementation of NGFP and CGFP enabled formation of GFP fluorescence. However, the similar GFP fluorescence was not monitored for YopM and LcrV.