Clone And Expression Of Pediocin PedA Gene From Pediococcus Acidilactici

As one of natural biopreservatives, bacteriocin plays important role in improvingfood safety and extending shelf-life. This research subject made use of geneengineering technology to clone the pediocin pedA gene from Pediococcusacidilactici and construct pediocin pedA gene expression system in Escherichia coliin order to solve the low bacteriocin production problem in wild strain and realizeexpected highly efficient expression of pediocin.The total DNA of P. acidilactici was used as the template to amplify thestructural gene pedA in the method of polymerase chain reaction (PCR). Theamplified pediocin structural gene pedA was ligased with pMD18-T vector beforetransformed E. coli DH5α competent cells. The pMD18-T recombinant plasmidcontaining pedA gene was extracted and digested by Bgl IIå'ŒXhol I restrictionenzymes and then ligased with the expression vector pET32a(+) which was digestedwith the same restriction enzymes. The ligation mixture was used to transform E. coliDH5α competent cells and further E. coli BL21(DE3) competent cells. Therecombinant strains were screened by PCR method and one was selected to extractrecombinant plasmid for DNA sequencing of the pedA gene. The result testified thatthe recombinant plasmid contained the pedA gene which was100%homologous tothe pedA gene published in the NCBI (GenBank AY083244). This recombinant strainwas induced with1mmol/L IPTG at37℃for4h and efficiently expressed22kDaTrx-Ped Afusion protein.The culture of the recombinant was sonicated and centrifuged. Both thesupernatant and the pellet were analized by SDS-PAGE. Since the result showed thatthe expected fusion protein was in the pellet, the protein was expressed as inclusionbody. To obtain active expression product, the extracted inclusion body was washedadequately and treated with the renaturation buffer containing GSSG-GSH system forthe correct refolding of the protein. The refolding protein took advantage of its ownHis-tag and was separated and purified by adding to the Ni-IDA agarose resin column.A single protein band was observed in500mmol/L imidazol elute. The purified fusionprotein has the concentration of0.4-0.7mg/mL. Purified fusion protein was treatedwith enterokinase and two protein bands appeared. One of them corresponded withthe predicted molecular mass of pedA gene; the other corresponded with the predicted molecular mass of thioredoxin.Purified fusion protein was treated with enterokinase and the well test revealedthat the cleavage product had antimicrobial activity to Listeria monocytogenes whichproved that the constructed pediocin pedA gene expression system in E. coli couldexpress pediocin fusion protein and obtain biological active pediocin product afterinclusion body renaturation and enterokinase treatment of the fusion protein. The welltest evaluated the inhibitory spectrum of the genetic engineering pediocin. The resultsindicated that the antimicrobial activity on and Lactobacillus plantarum as well as L.monocytogenes was found clearly and the antimicrobial activity againstStaphylococcus aureus, Salmonella typhi and E. coli O157was not found.