title: Recombinant antimicrobial peptide hPAB-β expressed in Pichia pastoris, a potential agent active against methicillin-resistant Staphylococcus aureus.
abstract: As a potential therapeutic agent, antimicrobial peptide has received increased attention in recent years. However, high-level expression of a small peptide with antimicrobial activity is still a challenging task. In this study, the coding sequence of antimicrobial peptide hPAB-β, a variant derived from human beta-defensin 2, was cloned into pPIC9K vector and transformed into Pichia pastoris. P. pastoris transformants harbored with multi-copy plasmids were screened by G418 selection. When the transformed cells were induced by methanol, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blot, and matrix-assisted laser desorption ionization-time of flight mass spectrometry revealed recombinant hPAB-β products consisting of three protein species of 4,680.4, 4,485.3, and 4,881.9 Da at proportions of 58%, 36%, and 6%, respectively, which may be due to the incomplete processing of the fusion signal peptide of α-factor by the STE13 protease. Expressed hPAB-β was secreted into the culture medium at a level of 241.2 ± 29.5 mg/L. Purified hPAB-β with 95% homogeneity was obtained by 10 kDa membrane filtration followed by cation ion-exchange chromatography with a SP-Sepharose XL column. The two major protein species separated through a SOURCE 30RPC reverse phase chromatography column showed definite antimicrobial activities against Staphylococcus aureus. All 22 methicillin-resistant S. aureus (MRSA) isolates with multidrug resistance phenotype were sensitive to the recombinant hPAB-β with minimal inhibitory concentrations of 8-64 μg/ml. Our results show that the methylotrophic yeast-inducible system is suitable for high-level expression of active hPAB-β, and that expressed hPAB-β in P. pastoris may be a potential antimicrobial agent against MRSA infection.
authors.affiliation: Department of Microbiology, College of Medicine, Key Lab of Microbial Engineering Under the Educational Committee in Chongqing, Third Military Medical University, Chongqing 400038, People's Republic of China.
authors.forename: Zhijin
authors.initials: Z
authors.lastname: Chen
authors.forename: Dongmei
authors.initials: D
authors.lastname: Wang
authors.forename: Yanguang
authors.initials: Y
authors.lastname: Cong
authors.forename: Jing
authors.initials: J
authors.lastname: Wang
authors.forename: Junmin
authors.initials: J
authors.lastname: Zhu
authors.forename: Jie
authors.initials: J
authors.lastname: Yang
authors.forename: Zhen
authors.initials: Z
authors.lastname: Hu
authors.forename: Xiaomei
authors.initials: X
authors.lastname: Hu
authors.forename: Yinling
authors.initials: Y
authors.lastname: Tan
authors.forename: Fuquan
authors.initials: F
authors.lastname: Hu
authors.forename: Xiancai
authors.initials: X
authors.lastname: Rao
mesh_terms: D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*; D001483:Base Sequence; D015870:Gene Expression; D006801:Humans; D055624:Methicillin-Resistant Staphylococcus aureus / Q000187:drug effects*; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D023083:beta-Defensins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*
chemical_list: D000900:Anti-Bacterial Agents; D011994:Recombinant Proteins; D023083:beta-Defensins