Exploration Of Optimal Expression Conditions In Lactobacillus Casei With PPG Vector

Comparison with the E.coli's expression system, the expression system of Lactic acidbacteria(LAB) is immature. It mainly reflects on the followings: low expression level ofheterologous protein, some genetic character's ambiguity, troublesome operation, instability ofexpression condition and so on. Therefore, explorating suitable expression conditions of LABvectors to improve expression level and establish the platform of LAB's expression was veryimportant. It has practical significance on the heterologous protein realizing its function.The experiment explored the ideal expression conditions of plasmid pPG. Escherichia coliβ-glucuronidase (gusA) gene was used as a reporter gene for analyzing expression conditions inLactobacillus casei 393. According to the critical factors during expression, we have explored thefollowing aspects: inducer, concentration of inducer, induction time, pH of MRS and incubationmode. The expression level of gusA could achieve 14.0ï¼…of the whole cell proteins when theactivated recombinant L.casei 393 was incubated in basal MRS medium of pH between 6.0 and 7.0,supplemented with 1ï¼…lactose, at 30℃or 32℃, without shaking, about 7 to 8h, to the absorbanceof A₅₉₀OD reaching about 0.5. The experiment has realized gusA's highly effective and stableexpression. Recombinant Lactobacillus casei with pPG-2 mediating secretion expression wasinduced by lactose and its supernatants were concentrated 50-fold under vacuum or by dialysis at4^C. But purpose protein couldn't be detected by West blot. However, an obvious protein bandcorresponding to gusA was detected by West blot in protein extracts, which shows that gusAremained attached to the cell surface. The glucose in MRS inhibits gusA's expression. Whenglucose's concentration was under 0.1ï¼…, expression of gusA was hardly affected; however, whenthe level of glucose achieved 0.5ï¼…, gusA's expression was badly repressed. As the content ofglucose reached 2ï¼…, gusA could't be seen. This experiment provides the basis for expressing otheractive protein (peptide) by exploring optimal expression conditions of plasmid pPG inLactobacillus.