Recombinant production of human β-defensin-2 protein in Pichia pastoris

Antimicrobial peptides (AMP) are short peptides containing 6-100 amino acids. It can be produced by living organisms, generally rich in cationic, hydrophobic, lysine and arginine amino acids. Human beta defensin-2 (hBD-2) is an antimicrobial peptide with low molecular weight, containing cysteine-rich cationic properties, and whose production is increased in case of infection in epithelial cells. Pichia pastoris is an expression system that can be used for eukaryotic protein production with advantages such as high expression, inducibility, and glycosylation mechanism close to eukaryotes. In this study, FaDu (ATCC® HTB-43™) cell line was induced by Pseudomonas aeruginosa (ATCC 10145) and RNA was isolated. cDNA was synthesized from these RNAs and hBD-2 was amplified with polymerase chain reaction using unique primers. The resulting hBD-2 was first subcloned into the pGEM-T easy vector. Then, it was cloned into the pPICZαA vector and transformed into P. pastoris X-33 cells by electroporation. Production of hBD-2 peptide in the obtained recombinant P. pastoris under the control of AOX promoter for 96 hours at 30 °C was monitored using agar diffusion test, qRT-PCR, SDS-PAGE and western blotting methods. As a result, it was determined that the hBD-2 peptide was produced at 30 ℃, 150 rpm and 96 hours with the induction of 2% methanol. The resulting functional active hBD-2 peptide can be used for therapeutic purposes.