Study On Recombinant Expression Of Human β Defensin 3 And Plant Des-pGlul-Brazzein

Food safety is one of the hot spot all the time, and the natural & safe food additive is the key to assuring food safety. Human p-defensin-3 (hBD₃) is expected to be developed as a new food antimicrobial additive because of its broad-spectrum antimicrobial activity and low cytotoxicity to human. The sweet-tasting protein des-pGlul-Brazzein, isolated from the fruit of Pentadiplandra brazzeana Baillion, is expected to be developed as a new food sweet additive because of its high sweetness and heat & pH-stability. The project aimed at: (1) High-level expression of hBD₃, des-pGlul-Brazzein and hBD₃-Bra mosaic gene in E.coli and Pichia pastoris respectively; (2) Optimizing the fermentation conditions of the target strains by flask shake. The main results are shown as following:According to Escherichia and yeast biased codon, the coding sequence of hBD₃, des-pGlul-Brazzein and mosaic gene hBD₃-Bra ( connected with the sequence coding thrombin cleavage site) were constructed respectively, and were inserted into the multiclone site in pET30a and pPIC9K vector respectively, resulting in the recombinant expression vectors pET-hBD₃, pEl-Bra, pET-hBD₃-Bra and pPIC9K-hBD₃, pPlC9K-Bra, pPIC9K-hBD₃-Bra.The fusion protein of hBD₃ was expressed by using pET30a as expression vector and E.coli as host, the recombinant protein was expressed after induction by IPTG, which accounted for 30.9% of total protein of host cell. The recombinant fusion protein of hBD3 was purified by affinity column and then digested by enterokinase and purified by affinity column again, resulting in the recombinant natural hBD₃. The two interested protein both have high antimicrobial activity against E.coli and S.aureus, similar as natural protein.The fusion protein of des-pGlul-Brazzein was expressed by using pET30a as expression vector and E.coli as host, the recombinant protein was expressed after induction by IPTG, which accounted for 35% of total protein of host cell. The recombinant fusion protein of des-pGlul-Brazzein was purified by affinity column and then digested by enterokinase and purified by affinity column again, resulting in the recombinant natural des-pGlul-Brazzein. The sweetness of the two interested protein is 200 times and 600 times than that of sucrose respectively, including kept after 4h at 80 °C.The chimeric protein of hBD3-Bra was expressed by using pET30a as expression vector and E.coli as host, the recombinant chimeric protein of hBD3-Bra was expressed after induction by IPTG, which accounted for 30.5% of total protein of host cell. 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 recombinant hBD3 has a high antimicrobial activity, and the natural des-pGlul-Brazzein also has 600-time sweetness of sucrose, much sweeter than that of fusion protein Most of the expressed proteins were in inclusion bodies, some was soluble. Both the soluble and renaturation protein from inclusion bodies have their own activity whether by dialysis or by column.The fermentation conditions for BL21-pET-hBD₃, BL21-pET-Bra and BL2l-pET-hBD3-Bra were optimized respectively by flask shake. The best conditions for growth and fermentation were basically same, i.e. culture volume 100mL (500mL flask), seed dose 1.5%, medium original pH value 7.0. The culture volume was the most important factor. The growth speed would be lower as the culture volume increased more. It suggested the content of soluble O2 is key factor for growth and fermentation of the strains,. The biomass would increase as time passed.In order to study the influence of different concentration of inducer, temperature and time on the growth and the yield of target protein of strain respectively, the strains were induced by IPTG and lactose at 37°C and 30°C for 4h and 6h respectively. The results showed that the concentration of IPTG had no obvious effect on the growth and the yield of expression of strains between 0.2-lmM, The concentration of lactose obviously inhibit the growth of s...