| Plectasin is the first defensin-type antimicrobial peptide isolated from a fungus, the saprophytic ascomycete Pseudoplectania nigrella. The mature peptide of Plectasin is a 40-residue carboxy-terminal domain, and its molecular weight is about 4.4 KDa. Plectasin contains a CSa(3 motif that is comprised of an a-helix and two antiparallel P-strands, and is stabilized by three disulphide bonds. Plectasin has potent activity against Gram-positive bacteria and is especially active against Streptococcus pneumoniae. Since plectasin shows strong antimicrobial activity with no hemolytic activity and extremely low toxicity, it becomes a novel defensin with therapeutic potential.The goal of this research was to develop an efficient gene system in E. coli for the production of a recombinant plectasin with high yield and activity in low cost by genetic engineering and to optimize the conditions of immobilized-metal affinity chromatography for fusion protein purification. According to the sequence of plectasin gene published in Gene Bank, the DNA fragment coding for the mature peptide of plectasin was synthesized and cloned in plasmid pPIC-9K. To construct an expression vector in E. coli, gene-specific PCR primers for the mature peptide of 40 amino acid residues were synthetized. The plectasin gene fragment in pPIC-9K was amplified by PCR and ligated into pET-32a(+) expressing vector. After sequencing, the pET-32a(+)-Plectasin expression vector was delivered into E. coli Rosetta(DE3) cells. The results showed that the levels of the Trx-Plectasin fusion protein produced in selected transformants reached over 50% of the total protein in E. coli, which showed a molecular mass of-25 KDa. After cell disruption, fusion protein Trx-Plectasin was purified by the immobilized-metal affinity chromatography method. The recombinant plectasin was effectively released by the treatment of the fusion protein with enterokinase and showed potent anti-Staphylococcus aureus activity. |
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