[Optimizing conditions for the expression of human beta defensin 3 and des-pGlu1-Brazzein in Escherichia coli and analysis of their activity].
The inductive conditions for the flask-shaking of E.coli BL21-pET-hBD3-Bra had been optimized, at the same time, the expressed protein had been purified and analyzed. The effect of three factors which were IPTG concentration, induction time and temperature on growth of strain and on the yield of hBD3-Bra was analyzed in detail. The result indicated that the concentration of IPTG had little effect on the growth and the expression of target protein between 0.2-1 mmol/L, Biomass would be improved as time passed, but the target protein didn't increase obviously as the same time, temperature was the most important factor, the expressed level of hBD3-Bra, as high as about 35% of total cell protein, could be gained when strain was induced by IPTG under 30 degrees C. Further analysis showed the best temperature for growth was 30 degrees C-32 degrees C and for expression protein was 30 degrees C.The purified hBD3-Bra has a weak antimicrobial activity, but is 200 times sweeter than that of sucrose. After digested by thrombin and purified by affinity column, the natural des-pGlul-Brazzein also has 600-time sweetness of sucrose, and the recombinant hBD3 has a high antimicrobial activity again E. coli and S. aureus.
Li
Chunli
C
College of Animal and Veterinary Science, Henan Agricultural University, Zhengzhou 450002, China.
Xu
Xueli
X
Zheng
Zhenyu
Z
Zhao
Weidong
W
D000890:Anti-Infective Agents; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D010940:Plant Proteins / Q000096:biosynthesis* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D013549:Sweetening Agents; D013696:Temperature; D023083:beta-Defensins / Q000096:biosynthesis* / Q000235:genetics
D000890:Anti-Infective Agents; C422950:DEFB103A protein, human; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D013549:Sweetening Agents; D023083:beta-Defensins; C090521:brazzein protein, Pentadiplandra brazzeana