Studies On The Expression And Purification Of Human Beta Defensins

Human defensins belong to the family of antimicrobial peptides, and play important roles in the innate human immune system. Human defensins have a broad antimicrobial spectrum and microbes are not easy to show resistance to this kind of antibiotics due to their special antimicrobial mechanism, so they are very promising in medical application.In this thesis, by using the codon-optimized strain of human beta defensin 4, BL21(DE3)/ pET32-smHBD4, the cultivation conditions were optimized considering the induction temperature, inducer concentration, induction timing and induction time in shake flask. The optimized culture conditions were: induction temperature 34℃, IPTG concentration 0.4mM, induction timing mid-exponential growth phase, induction time 6 hours. After optimization, the target fusion protein TrxA-HBD4 achieved 2.68g/l (mature HBD4 0.69g/l) and the percentage of target soluble fusion protein achieved 65% of the total soluble protein.Then, the thesis did some research in the separation and purification of human beta defensin 4 (HBD4). After a process of Ni affinity chromatography, enterokinase digestion, cationic ion exchange chromatography, gel chromatography desaltation and freeze drying, the recombinant HBD4 with a purity of 95% was obtained. And the recovery rate of the whole process was 53%.After that, the antimicrobial activity of recombinant HBD4 was studied. By inhibitory zone test, the recombinant HBD4 showed obvious antimicrobial activity against P. aeruginosa ATCC27853 and E. coli K12 D31. By liquid culture, the relationship between the HBD4 concentration and the antimicrobial activity against E. coli K12 D31 are tested and an inhibitory curve was drawn.In addition, by using the codon-optimized strain of human beta defensin 3, BL21(DE3)/ pET32-smHBD3, the cultivation conditions of human beta defesin 3 were optimized including the induction temperature, inducer concentration , induction timing, induction time in shake flask. The optimized culture conditions were:induction temperature 34 °C , IPTG concentration 0.4mM, induction timing mid-exponential growth phase, induction time 8 hours. After optimization, the target fusion protein TrxA-HBD3 achieved 2.55g/l (mature HBD3 0.57g/l) and the percentage of target soluble fusion protein achieved 66% of the total soluble protein.