The Function Of E.Coli YggG And Its Interactional Factor

Up to now, there is little report about the function of E.coli YggG protein which is a putative Era-binding protein found in E.coli by screening E.coli genome phage expression library. Sequence homologous analysis shows that YggG protein is a putative metalloprotease, but it hasn't been proved by experiments. Previous studies showed that over expression of YggG (294aa) protein could inhibit the growth of host bacteria, however, there are several ATG in the sequence of yggG gene, some of which can start translation and form different peptides. At the same time, there is an evident difference between the sequence of .yggG gene published by GenBank and SWISS-PROT. Therefore, the expression characteristic of YggG in E.coli is the first problem to be studied and confirmed.This study is to work out three problems: First, study the relationship between YggG proteins of different length translated from different ATG and their biological effects; Second, identify the expressive and distributive characteristic of YggG protein in E.coli cell; Third, to prove the relationship between YggG and Era protein.In order to elucidate above problems, the study is divided intothree parts:First, study the relationship between YggG proteins of different length translated from different ATG and their biological effects. In order to elucidate this problem, we identified the regions of YggG protein that could restrain the growth of E.coli and to acquire atoxic YggG mutants to study the effects of different YggG.Different length sequences of yggG gene were amplified by PCR and cloned into the same expression vector. The expression engineering bacteria of truncated YggG was constructed. The expressed YggG proteins of different length were induced under the same conditions, Then studied the cell growth curve, cell viability and the morphological changes of host bacteria bearing different truncated YggG protein by microscope and electron microscope. With the same methods ,we studied the effect of HEXXH motif of YggG on the toxic function by mutating two amino acide sites in HEXXH motif which is high homology in metalloprotease family. The results demonstrated that YggG protein of different length tanslated from different ATG had different biological effects, YggG protein that could inhibit the growth of bacteria contain YggG(294aa) , YggGM(294aa) , YggG(270aa, YggG(252aa) and YggG(42aa). However, YggG(231aa) and YggG(24aa) peptides did not affect the viability of host bacteria. We conclude that the integrity of sequence of YggG, spanning the amino-acid residues between w25-63 from the N-terminus, is required for toxicity, the toxic function of YggG protein was not influenced by the point mutation of HEXXH motif that formed a metal binding site.Second, analyzed the expressive and distributive characteristic of YggG protein in wild type Escherichia coli. Antiserum was preparedby immunizing rabbit with truncated YggG(231aa) protein which unlike the toxic YggG and could be overexpressed and prepared. The rabbit anti-YggG antiserum was further purified by binding with the whole proteins of E.coli cell, The liter and specificity of polyclonal antibody were detected by Western blotting; Then we detect the characterisric of YggG proteins expressed in Escherichia coli by Western blotting and immunofluorescence technology. The results showed that all toxic YggG proteins located in the cell membrane, cell wall or the interspace; The molecular weight of YggG protein expressed in wild type Escherichia coli is near YggG(231aa), which was also ture when YggG proteins expressed in some stroke states. The expression of E.coliYggG protein could be deleted in the level of sensitivity over10-14g. We conclude that both YggG(294aa) and YggG(252aa) proteins are not expressed in wild type Escherichia coli; YggG protein expressed in wild type bacteria may be YggG(231aa) or YggG(243aa). The expression level of E.coliYggG protein is very low, which suggests that YggG is a regulation protein rather than a stucture protein. The results also demonstrated that the over expression o...