Studies On Expression Strategies Of Recombinant Human β-Defensins

Human |3-defensin is a family of cationic antibiotic peptides with broad antibacterial spectrum discovered in recent years, which play important roles in the defense against microbial invasion.In this dissertation, with human (3-defensin 2 (HBD-2) as the representative molecule, a process of recombinant expression of soluble human (3-defensins was proposed, which includes the cloning and the codon optimization of the gene, the construction of expression vectors, the comparison and selection of expression systems, the optimization and scaling up of the expression, and the purification and bioactivity determination of the product. HBD-3 and HBD-4 were also expressed with the established process.Firstly, the expression performance was compared between two different HBD-2 genes. One was cloned by RT-PCR from human skin, and the other was synthesized chemically with preferential codons of hosts. The results revealed that, the expression level of target protein from the synthesized gene was 9 fold of the cloned one.Secondly, the selection of expression systems was made among E. coli, P. pastoris, and S. bacillus. Because of its performance of high-level expression of soluble target protein, a recombinant strain of E.coli was adopted as the HBD-2 production strain.Thirdly, the optimal conditions were determined as following: cultivation at 28 癈 in MBL medium, induction at middle stage of exponential growth with O.SmM IPTG, and post-induction expression for 8 h. A percentage of 92.3% of the target fusion protein was expressed in soluble form and the volumetric productivity reached 1.3 g/1. The expression vector was further improved to achieve soluble expression at 37 癈. The culture process was then scaled up in a 10 1 fermentor.Fourthly, a purification procedure was established to obtain the mature recombinant HBD-2 in a purity of 95.0% with a recovery of 29.2%. The antimicrobial activity of the product was also determined.At last, the same expression strategy was used for the expressions of HBD-3 and HBD-4 in E.coli. The products were produced in 37 ℃ with solubilities of 90.8% and 89.1%, and expression levels reached 33.6% and 51.3% of total cell protein respectively.