Efficient Production Of Soluble Human Beta-defensin In Recombinant E.coli Systems

Human beta-defensin-2 (HBD-2) is a cysteine-rich cationic 41 amino acid antimicrobial peptide with three disulphide bonds. It exhibits a broad range of antimicrobial activity, especially against gram-negative bacteria and yeast. Since it seems difficult for pathogenic microorganisms to acquire resistance against it, HBD-2 has a great potential in therapeutic usage. The objective of the thesis is to achieve high-level production of HBD-2 in recombinant E.coli. The research work includes construction and screening of recombinant expression systems of HBD-2, optimization of fermentation conditions, rudimental antibacterial assay, and recombinant expression of HBD-3 and HBD-4.Firstly, based on expression systems including pET-28a(+), pQE30, pBV220, pGEX-4T-2, pET-32a(+) and pMAL-p2, a series of expression vectors were constructed with synthetic coding sequence of HBD-2 precursor (using a method called codon optimization) or its tadem repeats as heterologous gene. In light of the comparison in expression level and solubility of product, pET32-shBD2, which contains coding sequence of TRX as fusion partner, was selected as the optimal expression vector. To explain the difference in expression among vectors containing different multiple copies of shBD2 genes, the effect of tandem repeats on the growth of recombinant strains and plasmid stability was examined. It was found that recombinant strains containing fewer copies of shBD2 genes showed obvious advantage in growth and plasmid stability, and the existence of glucose in culture medium brought about positive effect on the growth of host strains and plasmid stability while using expression vectors which contain more tandemly repeated shBD2 genes. Study on the expression of the cDNA of hBD2 and shBD2 in pET-32a(+) system revealed that preferential codons could dramatically enhance the expression of HBD-2 in E.coli.Then further optimization of fermentation conditions in shake flask using E.coli BL21(DE3)/pET32-shBD2 was carried out. An optimal culture medium, MBL, was chosen by employing comparative tests of three different media. It was found that at lower temperature the solubility of target protein could be greatly enhanced, and at 28 癈 the expression of soluble target protein and fermentation time were both optimum. The optimal medium volume in flask was 12% (v/v). Induction conditions are also important parameters in HBD-2 fusion expression. The maximum HBD-2 production was attained when the induction was done at the middle logarithmic phase with theaddition of 0.8mM IPTG, and the optimal post-induction time was 8 hours. On the optimal fermentation conditions, almost all of target fusion protein was expressed in soluble form (>92.3%), and the volumetic productivity of soluble fusion protein reached 1.3g/L. Then this fermentation system was successfully scaled up in a 10L bench-top fermentor.The final objective of study on the recombinant expression is to obtain a large amount of product with biological activity. The expressed fusion protein was purified and incised, and then mature recombinant HBD-2 was subjected to the antimicrobial assay using E.coli K12 D31 as the sensitive strain. It was comfirmed that recombinant HBD-2 exhibited obvious antimicrobial activity against E.coli K12 D31. Human beta-defensin-3 (HBD-3) and human beta-defensin-4 (HBD-4) belonged to the same family, human beta-defensin, as HBD-2. Expression vectors, pET32-smhBD3 and pET32-smhBD4, were constructed using optimized coding sequences of mature HBD-3 and HBD-4 as target genes, respectively. HBD-3 and HBD-4 were successfully expressed in E.coli, and percents of target proteins in total cellular protein reached 33.6% and 51.3% respectively with high solubility (ca. 90%) of both proteins. It was also found that if mature HBD-2 was directly fused to the fusion partner TRX, fusion product could be expressed efficiently with high solubility even at 37C.