Construction of human antimicrobial peptide LL-37 vector and its expression in Pichia pastoris / 第二军医大学学报

ABSTRACT

Objective: To construct a eukaryotic expression vector of human antimicrobial peptide LL-37 (pPIC9-LL-37) and to express it in P. pastoris. Methods: The full-length gene encoding antimicrobial peptide LL-37 was synthesized by overlap extension-PCR method using the sequence of LL-37 and P. pastoris biased codon. The full-length gene was cloned into pPIC9 vector and the product was transformed into E. coli DH5α to construct expression vector pPIC9-LL-37. After identification by PCR and sequencing, pPIC9-LL-37 was used to transfect P. pastoris. The expression of LL-37 was induced by methanol and the highest expressing strain was screened. The concentrated fermentation product was analyzed by Tricine-SDS-PAGE and Western-blot, and the antibacterial activity of the expression product on E. coli. DH5α was tested. Results: The eukaryotic expression vector pPIC9-LL-37 was successfully constructed. The fusion of LL-37 gene into P. Pastoris was confirmed by PCR. High expression of LL-37 was expressed by 0.5% methanol and the highest expressing strain was screened out. The fermentation supernatant contained 0.5 μg/ml LL-37 and had strong antibacterial activity against E. coli. Tricine-SDS-PAGE and Western-blot analysis confirmed that the product was LL-37. Conclusion: We have successfully constructed pPIC9-LL-37 for transfecting P. pastoris. Methanol can induce the high expression of LL-37 and the expressed LL-37 has strong antimicrobial activity.