The Expression Of Antibacterial Peptide DCD-1L By Random Mutated In Pichia Pastoris

To following of the wide application of antibiotics, homologous, the bacterial resistance of conventional antibiotics also emerged more seriously. In this urgent we found a new way of preparation of antimicrobial with higher activity and broader- spectrum for drug territory. Dermcidin is a new antibacterial peptide recently found from human sweat glands, and DCD-1L is a derivant of Dermcidin which has a broad- spectrum antimicrobial peptide. It is a new class of antibacterial medicine having enormous potentiality. Therefore it has great economic value and theoretical significance to make random mutations in gene with the method of directed evolution and to express in Pichia pastoris for screening an antibacterial peptide with even higher activityIn this research, the gene with the sequence of random mutations by overlap extention PCR and it has about 167 bp through mutation causing by oligonucleotide in orthogenesis. The eukaryotic expression system with tacho-development and most potency was choiced, the gene was inserted into the Pichia pastoris expression vector pGAPZαC Which contains GAP promotor andα-factor signal sequence. The recombinant vector was integrated in Pichia pastoris by electroporation,and which formed a mutation library with the capacity of 1. 09×10~4. The multicopied recombinant Pichia strains were obtained by screening of Zeocin resistance. They were identified by PCR and expressed. SDS-PAGE analysis showed that the genes were expressed, and the proteins were secreted into the medium andwere about 6000D which was the reason of the proteins by moderate modification ofglycosylation. In this study, we have constructed a mutation library in Pichia pastoris expression vector pGAPZαC by the method of directed revolution, and achieved expression of random-mutated DCD-1L in eukaryotic system.