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| Expression of Channel Catfish (Ictalurus punctatus) Hepcidin Mature Peptide in Pichia pastoris and Its Antibacterial Activity |
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Abstract Hepcidin is a kind of small cysteine-rich cationic antimicrobial peptide which mainly expressed in livers of animals, and play an important role in immune response against microbial invasion. Thus, it is considered to be good substitutes for traditional antibiotics. In the present study, the gene encoding channel catfish (Ictalurus punctatus) hepcidin mature peptide (mCH) was synthesized with reference to its native DNA sequence according to preference of Pichia pastoris. EcoRⅠand NotⅠrestriction sites were added to 5' and 3' ends of the mCH by PCR, respectively. The expected fragment was ligated with pPIC9K vector to construct a recombinant expression vector pPIC9K-mCH which then was transformed into competent Pichia pastoris GS115 cells. The transformants (His+Mut+) containing multicopy gene insertion were screened with increasing concentration of antibiotic G418. Recombinant mCH was induced with 1.0% (V/V) methanol at 30 ℃, pH 6.0 for different times. The results showed that channel catfish mCH consisted of 25 residues, and 8 conserved cysteine residues were located at its C-terminal, suggesting that this region was responsible for its antibacterial activity. The total protein in the supernatant culture reached a peak concentration at 72 h during the induction period, which indicated that 72 h was an optimal expression time. Furthermore, the recombinant mCH was purified by SP Sepharose Fast Flow column with 20 mmol/L PB (pH 6.5) containing 500 mmol/L NaCl. Tricine-SDS-PAGE analysis demonstrated that molecular mass of the purified recombinant mCH was 3 800 D. According to the concentration of purified mCH, expression yield of the recombinant mCH was calculated to be 51 mg/L. Antibacterial assays showed that the fermentation supernatant concentrated for 10 and 15 times containing the recombinant mCH had bioactivities against Staphylococcus aureus and Escherichia coli. The present study first realized the recombinant DNA expression of channel catfish hepcidin mature peptide in Pichia pastoris, which provides the important initial value for industrial production of hepcidin antimicrobial peptide.
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Received: 01 September 2014
Published: 02 February 2015
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| [1]Ahmad M, Hirz M, Pichler H, et al.Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous protein production[J]., 2014, :-
[2]Bao BL, Peatman E, Li P, et al.Catfish hepcidin gene is expressed in a wide range of tissues and exhibits tissue-specific upregulation after bacterial infection[J].Developmental and Comparative Immunology, 2005, (29):939-950
[3]Hunter HN, Fulton DB, Ganz T, et al.The solution structure of human hepcidin, a peptide hormone with antimicrobial activity that is involved in iron uptake and hereditary hemochromatosis[J].Journal of Biological Chemistry, 2002, (277):37597-37603
[4]Koliaraki V, Marinou M, Samiotaki M, et al.Iron regulatory and bactericidal properties of human recombinant hepcidin expressed in Pichia pastoris[J].Biochimie, 2008, (90):726-735
[5]Krause A, Neitz S, Mgert HJ, et al.LEAP-1, a novel highly disulfide-bonded human peptide, exhibits antimicrobial activity[J].FEBS Letters, 2000, (480):147-150
[6]Li L, Wang JX, Zhao XF et al.High level expression, purifcation, and characterization of the shrimp antimicrobial peptide, Ch-penaeidin, in Pichia pastoris[J].Protein Expression and Purification, 2005, (39):144-151
[7]Nicolas G, Viatte L, Bennoun M, et al.Hepcidin,a new iron regulatory peptide[J].Blood Cells, Molecules, and Diseases, 2002, 29(（3）NovDec):327-335
[8]Park CH, Valore EV, Waring AJ, et al.Hepcidin, a urinary antimicrobial peptide synthesized in the liver[J].Journal of Biological Chemistry, 2001, (276):7806-7810
[9]Rashid F, Ali I, Sajid I, et al.Recombinant expression of an antimicrobial peptide hepcidin in Pichia pastoris[J].Pakistan Journal of Zoology, 2009, 41(5):349-356
[10]Schagger H, Von Jagow G.Tricine-sodium dodecyl sulfatepolyacrylamide gel electrophoresis for the separation of protein in the range from 1 to 100 ku[J].Analytical Biochemistry, 1987, (166):368-379
[11]Tao Y, Zhao DM, Wen Y.Expression, purification and antibacterial activity of the channel catfish hepcidin mature peptide[J].Protein Expression and Purification, 2014, (94):73-78
[12]Yadava A, Ockenhouse CF.Effect of codon optimization on expression levels of a functionally folded malaria vaccine candidate in prokaryotic and eukaryotic expression systems[J].Infection and Immunity, 2003, 71(9):4961-4969
[13]Zhu WH, Sun MN, Wang YS, et al.Expression and functional characterization of a recombinant targeted toxin with an uPA cleavable linker in Pichia pastoris[J].Protein Expression and Purification, 2011, (76):184-189[J].Protein Expression and Purification, 2011, (76):184-189
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