Expression, purification and characterization of the recombinant cysteine-rich antimicrobial peptide snakin-1 in Pichia pastoris

• Published in: Protein expression and purification 2016-06, Vol.122, p.15-22
• Main Authors: Kuddus, Md. Ruhul, Rumi, Farhana, Tsutsumi, Motosuke, Takahashi, Rika, Yamano, Megumi, Kamiya, Masakatsu, Kikukawa, Takashi, Demura, Makoto, Aizawa, Tomoyasu
• Format: Article
• Language: English
• Subjects: Anti-Infective Agents - chemistry; Anti-Infective Agents - isolation & purification; Anti-Infective Agents - metabolism; Anti-Infective Agents - pharmacology; Antimicrobial peptide; Bacteria - drug effects; Bacterial Infections - drug therapy; Cloning, Molecular - methods; Cysteine-rich; Escherichia coli; Folding; Fungi - drug effects; Humans; Membrane permeability; Mycoses - drug therapy; Over-expression; Pichia - genetics; Pichia pastoris; Plant Proteins - chemistry; Plant Proteins - genetics; Plant Proteins - isolation & purification; Plant Proteins - pharmacology; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - pharmacology; Snakin-1; Solanum tuberosum; Solanum tuberosum - genetics; Transformation, Genetic
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2016.02.002

Snakin-1 (SN-1) is a small cysteine-rich plant antimicrobial peptide with broad spectrum antimicrobial activity which was isolated from potato (Solanum tuberosum). Here, we carried out the expression of a recombinant SN-1 in the methylotrophic yeast Pichia pastoris, along with its purification and characterization. A DNA fragment encoding the mature SN-1 was cloned into pPIC9 vector and introduced into P. pastoris. A large amount of pure recombinant SN-1 (approximately 40 mg/1L culture) was obtained from a fed-batch fermentation culture after purification with a cation exchange column followed by RP-HPLC. The identity of the recombinant SN-1 was verified by MALDI-TOF MS, CD and 1H NMR experiments. All these data strongly indicated that the recombinant SN-1 peptide had a folding with six disulfide bonds that was identical to the native SN-1. Our findings showed that SN-1 exhibited strong antimicrobial activity against test microorganisms and produced very weak hemolysis of mammalian erythrocytes. The mechanism of its antimicrobial action against Escherichia coli was investigated by both outer membrane permeability assay and cytoplasmic membrane depolarization assay. These assays demonstrated that SN-1 is a membrane-active antimicrobial peptide which can disrupt both outer and cytoplasmic membrane integrity. This is the first report on the recombinant expression and purification of a fully active SN-1 in P. pastoris. •We established a Pichia expression system for enhanced production of bioactive SN-1.•Six disulfide bridges were formed efficiently without a renaturation process.•About 40 mg of recombinant SN-1 was obtained from 1L fermentation culture.•NMR studies of recombinant SN-1 suggested the formation of correct disulfide bonds.•Purified recombinant SN-1 exhibited microbicidal activity by membrane disruption.