Random Mutations Of Antibacterial Peptide DCD-1L And Expression In Pichia Pastoris

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 of activities against Gram negative and positive bacteria. They are new class of antibacterial compounds having great potentialities. Therefore it has high 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 activity.The methylotrophic yeast Pichia pastoris is now one of the important tools used in molecular biology for the generation of recombinant protein. As yeast, Pichia pastoris is a single-cell microorganism that is easy to manipulate and culture. It is also a eukaryote and capable of the posttranslational modifications performed by higher eukaryotic cells, such as proteolytic processing, folding, disulfide bond formation, and glycosylation.The gene with the sequence of random mutations through splicing by overlap extension was inserted into the Pichia pastoris expression vector pGAPZ α C which contains GAP promotor and α -factor signal sequence. The recombinant Pichia strains were identified by PCR and Zeocin resistance. SDS-PAGE analysis showed that the genes were expressed and the proteins were secreted into the medium. Due to the limitation of LiCl transformation method, only 10~4 different recombinant clones were obtained. We have not screened interest antibacterial peptides so far which requires us to try out an improved method to make some achievement. In this study, we have constructed a mutation library in Pichia pastoris expression vector pGAPZ α C by the method of directed revolution, and achieved expression of random-mutated DCD-1L in eukaryotic system.