title: Expression, purification and characterization of the recombinant cysteine-rich antimicrobial peptide snakin-1 in Pichia pastoris.
abstract: 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 (1)H 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.
authors: Graduate School of Life Science, Hokkaido University, Sapporo, Hokkaido 060-0810, Japan; Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Dhaka, Dhaka 1000, Bangladesh.
authors: Md Ruhul
authors: MR
authors: Kuddus
authors: Graduate School of Life Science, Hokkaido University, Sapporo, Hokkaido 060-0810, Japan.
authors: Farhana
authors: F
authors: Rumi
authors: Graduate School of Life Science, Hokkaido University, Sapporo, Hokkaido 060-0810, Japan.
authors: Motosuke
authors: M
authors: Tsutsumi
authors: Graduate School of Life Science, Hokkaido University, Sapporo, Hokkaido 060-0810, Japan.
authors: Rika
authors: R
authors: Takahashi
authors: Graduate School of Life Science, Hokkaido University, Sapporo, Hokkaido 060-0810, Japan.
authors: Megumi
authors: M
authors: Yamano
authors: Graduate School of Life Science, Hokkaido University, Sapporo, Hokkaido 060-0810, Japan.
authors: Masakatsu
authors: M
authors: Kamiya
authors: Graduate School of Life Science, Hokkaido University, Sapporo, Hokkaido 060-0810, Japan.
authors: Takashi
authors: T
authors: Kikukawa
authors: Graduate School of Life Science, Hokkaido University, Sapporo, Hokkaido 060-0810, Japan.
authors: Makoto
authors: M
authors: Demura
authors: Graduate School of Life Science, Hokkaido University, Sapporo, Hokkaido 060-0810, Japan. Electronic address: aizawa@mail.sci.hokudai.ac.jp.
authors: Tomoyasu
authors: T
authors: Aizawa
mesh_terms: D000890:Anti-Infective Agents / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology*; D001419:Bacteria / Q000187:drug effects; D001424:Bacterial Infections / Q000188:drug therapy; D003001:Cloning, Molecular / Q000379:methods; D005658:Fungi / Q000187:drug effects; D006801:Humans; D009181:Mycoses / Q000188:drug therapy; D010843:Pichia / Q000235:genetics*; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D011198:Solanum tuberosum / Q000235:genetics*; D014170:Transformation, Genetic
chemical_list: D000890:Anti-Infective Agents; D010940:Plant Proteins; D011994:Recombinant Proteins; C117071:SN1 protein, Solanum tuberosum
keywords: Antimicrobial peptide; Cysteine-rich; Folding; Membrane permeability; Over-expression; Pichia pastoris; Snakin-1