Gallinacin-3: High-level Expression in Escherichia Coli and Refolding of the Recombinant Protein

Abstract: β -defensin Gallinacin-3 (Gal-3) is the most important member of a large family of antimicrobial peptides in chicken. Gal-3 displays a strong antimicrobial activity against bacteria and fungi. Furthermore, it also plays indispensable function in triggering adaptive immune responses. So it could act as a substitute for antibiotic. In this paper, systems for high-level expression of the β -defensin fusions in Escherichia coli were constructed, and expression regulation and the in vitro refolding of the recombinant β -defensin were investigated.There are three parts in this paper: ① Construction of prokaryotic expression vectors of chicken β -defensin The PCR-amplified Gal-3 cDNA coding for the mature form in-frame into was inserted into plasmids pGEX6p-1、 pQE30、 pET30a and pET22b, which could expression Gal3 fused with Glutathione-S-transferase (Gal3-GST) or with hexamer histidine (Gal3-His) in different forms. ② Induction of β -defensin expression by Escherichia coli After induction with isopropyl- β -D-thioGalactoside (IPTG). The Escherichia coli harboring pGal6p-l expression vector could express a novel protein (about 32kD), which is identical in size with the expected Gal3/GST. However, the Gal-3/His constructs could not express the aimed protein in E. coli no matter the position and number of His-peptide being fused in. The Gal-3/GST is expressed in insoluble form as inclusion body, and its yield was about 35% in the all bacteria protein. Top yield of recombinant Gal-3/GST could be achieved with as less as 0.1mmol/L IPTG induction after 2 hours. (3) In vitro refolding of recombinant β-defensin The inclusion body of Gal3/GST was dissolved in the buffer containing 8mol/L Urea, then diluted gradually into the refolding system which containing L-Glutathione and Reduced L-Glutathione. A bioassay was designed to evaluated the biological activity and protein recovery of the recombinant β -defensin. Accordingly, about 30% reconstruction protein was refolded perfectly. Furthermore, recombinant Gal3/GST could completely suppress the growth of Salmonella enteritidis and Enteropathogenic E.coli,ihe minimal bactericidal concentration was about 12 μ g/ml and 24 μ g/ml. The present data provide essential basis for industrial production of chicken β -defensin in the future…
Key words: Chicken β-defensin; gene recombinant; refolding; antimicrobial antivity

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