Preliminary Studies On Expression Of Bovine Tracheal Antimicrobial Pepetide In Pichia Pastoris

Bovine tracheal antimicrobial peptide (bTAP) is a peptide from bovine tracheal mucosa with a broad spectrum of antimicrobial activity. However, the source f natural bTAP is very limited. The methylltrophic Pichia pastoris yeast is a novel system for expressing recombinant proteins with easy manipulation and correct post-translational modifications such as proteolytic processing, folding, disulfide bond formation and glycosylation.In this research two pairs of primers were designed according to published bTAP gene sequence and the codon usage of Pichia Pastoris and the mature peptide-coding sequence was amplified by overlapping PCR. The gene fragment was cloned into Pichia Pastoris expression vector pPIC9K. After linearization by Sal I- or Bgl II digestion, the recombinant pPIC9K-bTAP was transformed into GS115 cells by electroporation. Three Mut+ yeast recombinants resistant to 3mg/ml G418 and one Muts recombinant resistant to 1mg/ml G418 were selected. The recombinant yeasts were re-transformed with pPIC9K-bTAP vector and even higher copies of homologous recombinants were obtained. SDS-PAGE of methanol-induced yeast cultures showed that a peptide of 4kDa was expressed with antimicrobial activity.