Recombinant Expression,Purification And Activity Of Antimicrobial Peptide,Mytichitin-A And Plantaricin ZJ5

Plantaricin ZJ5 is one of Bacteriocins isolated from fermented mustard with a broad range of inhibitory activity,containing a 22-amino-acid and the molecular mass of the peptide was 2572.9 Da.The antimicrobial peptide Mytichitin-A was isolated from the serum of Mytilus coruscus,which was an important mytilus on aquiculture.This peptide consisted 55 amino acids and had a 6621.55 Da with 6 Cysteines presented in its sequence forming 3 disulfide bonds.In our study,both of the Plantaricin ZJ5 and Mytichitin-A were cloned into pGEX-2T vector and pMAL-c2x vector respectively.Protein expression was induced with Isopropyl-thio-galactoside(IPTG).The yield of GST-PlantaricinZJ5 and GST-Mytichitin-A reached 113 mg/L and 84 mg/L after purification.And the Tag of fusion protein was removed by degesting with enterokinase.Unfortunately,the Plantaricin ZJ5 and Mytichitin-A showed no antimicrobial activity.Then we inserted the sequence encoding the Mytichitin-A gene into the pPICZaA vector.The SacI-linearized plasmid pPICZaA-Mytichitin-A was transformed into P.pastoris GS115 by electroporation.The recombinant Mytichitin-A was secreted into culture after induction of methanol.And 150 mg pure recombinant Mytichitin-A was obtained from 1 L culture by nickel ions affinity purification.The result of antimicrobial assays indicated that the recombinant mytichitin-A showed significant antimicrobial activity against S.Aureus(ATCC 25923)and B.Subtilis1511-1.In particular,the purified product has a minimum inhibition concentration(MIC)of 31 ?g/ml on S.aureus(ATCC 25923).Taken together,our data shown that rMytichitin-A derived from the serum of Mytilus coruscus can be expressed and secreted efficiently when Pichia pastoris was used as a cell factory.This is the first report on heterologous expression of Mytichitin-A in P.pastoris and the approach described holds great promise for application of antimicrobial peptide.