Dissertation > Biological Sciences > Molecular Biology > Genetic engineering (genetic engineering)

Efficient Production of Bioactive Human Beta-defensin and Bovine Enterokinase in Recombinant Escherichia Coli

Author HuangLei
Tutor ZuoPeiLin;XuZhiNan
School Zhejiang University
Course Biochemical Engineering
Keywords Humanβ-defensin Enterokinase Genetic engineering Optimization Refolding Purification
Type PhD thesis
Year 2006
Downloads 331
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Human β-defensins are a group of cationic antibiotic peptides with broad antibacterial spectrum discovered in recent years, which play important roles in the self-defense against microbial invasion. Enterokinase is a serine Proteinase of the intestinal brush border that exhibits specificity for the sequence (Asp)4-Lys and converts trypsinogen into its active form trypsin.In this dissertation, a process of recombinant expression of human β-defensin-3 and -4(HBD3, HBD4) as well as bovine enterokinase light chain (BEKLC) was proposed, which includes the codon optimization of the genes, the construction of expression vectors, the optimization of the expression conditions, the purification of the target protein, refolding the inclusion bodies of BEKLC and bioactivity determination of the product.The codon optimized sequences coding HBD3 and HBD4 gene were synthesized, which were fused with TrxA to construct the expression vector. The resulting vector was finally transformed into E. coli BL21 (DE3) for expression. The optimal culture conditions of HBD3 and HBD4 production strain were determined as following: cultivation at 28℃ in MBL medium, induction at middle stage of exponential growth with 0.4 mM IPTG, and post-induction expression for 8 h of HBD3 and 6 h of HBD4. The high level expressions of HBD3 and HBD4 fusion protein were realized and never reported before. The volumetric productivity of fusion proteins were 2.55 g/l and 2.68 g/l for HBD3 and HBD4, respectively.A codon optimized sequence coding light chain of bovine enterokinase gene (sBEKLC) was synthesized, and it was fused with DsbA to construct the expression vector. Then the plasmid was transformed into E. coli BL21 (DE3) for expression, the volumetric productivity of fusion protein reached 151.2 mg/L, i.e. 80.6mg/L sBEKLC. The cold osmotic shock technique was successfully used to extract sulale sBEKLC from periplasmic space, and nickel affinity chromatography was employed to obtain mature sBEKLC. Finally, about 6.8mg of sBEKLC was purified from 1 liter fermentation broth and the enzyme activity of sBEKLC was well demonstrated.The inclusion bodies of sBEKLC fusion protein were purified under denaturingconditions and refolded by using refolding buffer in Ni-NTA column. The optimal refolding conditions were determined as following: 50 mM Tris-HCl (pH 8.0), 0.3 M NaCl, 2 M urea, 10 mM Imidazole, 3 mM GSH, 0.6 mM GSSG and refolding for 6 h. The enzyme activity of the product was detected.The self-made sBEKLC was successfully applied to split fusion protein and to release the mature bioactive HBD3 or HBD4. The purification procedure was established to obtain the recombinant HBD3 and HBD4. The overall recovery ratio of HBD3 was 41% with a purity of 92%, and which of HBD4 reached 44% with a purity of 95%. The antimicrobial activitis of the products were well determined.

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