Proteomics Analysis Of B.S 168 Overproducing PGA

Bacillus subtilis(B.subtilis)expression system has obvious advantages over E.coli expression system. A comprehensive understanding of proteomic changes in B.subtilis overexpression exogenous protein and knowledge of its mechanism helps guide the reconstruct of B.subtilis expression system,serving the industry better. To date, the study of its mechanism to secretory stress is mainly still on the RNA level rather than the protein.pAcc+pUB110 is a E.coli-B. Subtilis shuttle vector constructed by this lab. It includes pga following Pamy encoding Penicillin G Acylase(PGA), which making B.S 168 can secretory large quantities of PGA in the logarithmic growth phase with the induction of starch. under this laboratory conditions, the quality of this recombinant PGA can reach 1.6 g/L,and 10 U/mL, increasing by 10 folds than huang,and 2 folds than yang.,which providing a good experimental material for the studying of secretory stress response.In order to reduce the loss of intracellular proteins, this paper uses relatively simple sample preparation method: resuspending the collected bacteria in lysate(which refered to Aberdeen and made some improvement), then assisted with sonication. By measuring the growth curves and activity curve of control(B.S168/ pUB110)and high expression of PGA bacteria(lB.S168/pAcc+pUB110), this paper decided prepareing at the time point of 24 h, and by protein concentration determination ,this paper decided 15OD in 500μL lysate to prepare 2-DE sample. The 2-DE pictures shows a good repeatability by software analysis. The result shows great difference in differentially expressed proteins with use of different strip of pH3-10NL and pH4-7. There are 55 upregulate proteins and 12 downregulate protein with the former strip ,while 18 upregulat proteins and 3 downregulate with the latter. With MALDI-TOF-TOF mass spectrometry analysis, we identify 6 proteins。Four of them are caused because of growth inhibition by PGA overexpression, while PhoR and YxiE are related to response to overexpression . This results provide new clues to understand the response to overexpression stress intracellular better.