The Function Research Of Accessory Protein Of Phospholipase A1 In Serratia Marcescens

Phospholipase A1 is an important class of organisms hydrolase which can hydrolyse phospholipids to generate lysophosphatide and free fatty acids and thus widely used in the industry, mainly in plant degumming, food industry, pharmaceutical indust. There is a protein in serratia marcescens, phospholipase A1 auxiliary protein PlaS, which is relevant of phospholipae activity、host bacterium growth protein and secretion. And plaS gene which locates in the downstream of plaA gene overlaps with plaA。The overlap section of the two genes belongs to the typical over lapping gene, called plaB gene. The PlaS and PlaA have closely relations on their gene and protein level. So the main aspects of this study are as following on research the interaction between PlaA and PlaS:(1)Fisrt, SDS-PAGE analysis of plaS-pET28a/BL21(SP28) recombinant constructed bacteria while no band were found. Band was present by Western Blot even the expression level was low.. That provides an important theoretical basis for the construction of fusion recombinant bacteria(2) To set a control, we successfully constructed egfp-pET28a/BL21 (EP28), egfp-plaS-pET28a/BL21 (ESP28), plaS-egfp-pET28a/BL21 (SEP28) recombinant bacterial stains, which different in the order of plaS and egfp gene. According to the optimum of inducible expression、growth curve and fluorescence intensity, the fusion gene got best expression when plaS gene located in the downstream of the fusion gene The fusion protein has no inhibition on the growth of recombinant bacteria and fluorescence was observed clearly..(3) FRET were operated to verify the interaction of PlaA and PlaS. PlaA-CFP-pWSK29/BL21 (ACP), YFP-plaS-pET28a/BL21 (YSP) and plaA-CFP-pWSK29-YFP-plaS-pET28a/BL21 (YSAC) recombinant bacterial stains were constructed. The optimum of inducible expression、growth curve and fluorescence intensity were analyzed. Only YSP has a specific band in SDS-PAGE. Fluorescence signal was detected in ACP、YSP and YSAC.According to the signal of YSAC on the exciting light of 436, PlaA has an interaction with PlaS.(4)Constructed plaA-pET28a/BL21(AP28)、plaB-pET28a/BL21 (BP28、plaS-pET28a/BL21(SP28) and P28 recombinant bacteria were induced and observed by electron microscopy (sem) and transmission electron microscope, the cells of AP28 and p28 groups was full and AP28 cells was dense. That may because of phospholipase Al accumulate in cells. BP28 and SP28 were damaged in the extent which may be caused by high-activity expression of phospholipaseA1 and PlaS protein itself.In this thesis, phospholipaseAl auxiliary protein plaS gene of Serrtia marcescens was low expressed in E.coli,.The fusion protein constructed was analyzed by FRET and the interaction of PlaS and PlaA was found. All above lay a foundation of the structural and mechanical research about PlaS.