title: Secretion and activity of antimicrobial peptide cecropin D expressed in Pichia pastoris.
abstract: To express the antimicrobial peptide cecropin D in Pichia pastoris and determine the activity of the expressed product, four oligonucleotide fragments were synthesized in accordance with the available cecropin D sequences and a codon bias suitable for Pichia pastoris. Sequence fragments were phosphorylated, annealed, linked and cloned into the expression vector pGAPZαA and the yeast α-mating factor signal peptide was used as the signal sequence. The P. pastoris SMD1168 cells were transformed by electroporation using the constructed recombinant plasmid pGAPZαA-cecropin D. We were able to demonstrate by PCR that the cecropin D sequence had integrated into the P. pastoris genome. The expressed and secreted product was identified using Tricine-SDS-PAGE. Antibacterial activity was demonstrated using an agarose diffusion test and turbidimetry. The molecular mass of the recombinant cecropin D was estimated to be 3,900 Da. The recombinant cecropin D exhibited antibacterial activity for both Gram-positive and Gram-negative bacteria, suggesting that cecropin D was successfully expressed in P. pastoris. This approach holds great promise for antibacterial drug development.
authors.affiliation: College of Veterinary Medicine, South China Agricultural University, Guangzhou, Guangdong 510642,  P.R. China.
authors.forename: Chunhe
authors.initials: C
authors.lastname: Guo
authors.forename: Yumao
authors.initials: Y
authors.lastname: Huang
authors.forename: Hongyu
authors.initials: H
authors.lastname: Zheng
authors.forename: Liyun
authors.initials: L
authors.lastname: Tang
authors.forename: Jun
authors.initials: J
authors.lastname: He
authors.forename: Linsheng
authors.initials: L
authors.lastname: Xiang
authors.forename: Dehui
authors.initials: D
authors.lastname: Liu
authors.forename: Houquan
authors.initials: H
authors.lastname: Jiang