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

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