Systematically Analysis Of Protein Secretion And Protein Secretion Expression Of Bacillus Thuringiensis

Bacillus thuringiensis?BT?and BT formulation are the world's most widely used biological pesticide.BT is a gram positive bacterium with one layer of lipid bilayer membrane,BT can produce large amounts of extracellular proteins,which makes it a potential production bacteria for secreting proteins.Currently,there are few studies on the purpose of constructing BT engineering bacterium.The following aspects were studied in the paper.Based on signalP 4.1,TatP 1.0 and LipoP 1.0,the signal peptides of all the proteins in Bt HD73 were analyzed,and subcellular location were analyzed by psorb 3.0.4.The amount of secretory proteins containing Sec signal peptides is more than those containing Tat signal peptides,and the amount of Lipo proteins is the least of these three types.Most of Lipoproteins also contains a Sec signal peptide.All the proteins containing signal peptides are classified as transporters,spore and fermentation related proteins,virulence proteins,environment adaption proteins,expression and regulation proteins,energy metabolism proteins,cell surface proteins and enzymes,most of which are secreted by Sec or Tat signal peptides.In order to reduce background proteins and secretion protein degradation when constructing Bt expression strain,secretion proteins like collagenase,peptidase,amylase,bacillolysin and PlcR regulon could be knocked out.The growth curves of Bt HD73?WT?and Bt HD73??PlcR?were drawn,both of their OD₆₀₀ when T0 are around 2.6,the population doubling time are 36 min and 39 min separately,the knocking out of PlcR regulon doesn't affect cell growth and cell morphology.Comparing the extracellular proteins produced by Bt HD73?WT?and Bt HD73??PlcR?,the knocking out of PlcR regulon results in the absence of virulence protein production.With growth time extended,the amounts and kinds of extracellular proteins reduce constantly.11kinds of most abundant extracellular proteins are determined by Q-TOF,which provides signal peptides information for high efficiency protein secretion.A plasmid pHT315-?A3-1661-his was constructed based on pHT315-1661-gfp.The newly constructed plasmid is a shuttling plasmid between E.coli and Bt,which is composed of composition promoter?A3,his tag and terminating sequence from pET21b,and two restriction sites BamH I and Sal I.The plasmid pHT315-?A3-1661-his containing HD73₁661 gene was transferred into Bt HD73?WT?,and protein production analysis,Ni chromatography and Western Blot were conducted to confirm the production of secretion protein with his tag.The signal peptides of all proteins in Bt HD73 were analyzed in the paper,proteins produced by Bt HD73?WT?and Bt HD73??PlcR?were compared,11 kinds of most abundant secretion protein were determined,a Bt HD73 secretion plasmid was constructed and its function was confirmed.All these provide information for secretion protein study of Bt and construction of high efficiency protein secretion Bt.