Purification And Characterization Of Pediocin Expressed By Escherichia Coli

Our laboratory have already constructed an E.coli BL21 (DE3) expression system contained a recombinant plasmid with Ped A gene which can express the Pediocin fusion protein efficiently. This research was mainly focus on the purification and characterization of pediocin expressed by Escherichia Coli.The research compared two methods——the IPTG induction and the auto-induction by ZYM-5052 media during the expression course of gene engineering E.coli BL21 (DE3). The experiments results showed that auto-induction was superior to IPTG induction. The final OD600 of the fermented medium in auto-induction was around 7, whereas only around 3 in the IPTG induction, and the protein concentration of the dissolved inclusion body in auto-induction was 0.2359mg/ml, whereas only 0.1001mg/ml in IPTG induction.Considering the high expression of gene engineering E.coli BL21 (DE3), the pediocin fusion protein always existed by the formation of inclusion body(IB). The research tested several strategies of the refolding of IBs including the direct dilution, reversed dilution, dialysis, CTAB assisted and CTAB andβ-CD assisted refolding. The results figured that CTAB andβ-CD refolding which succeeded to keep a high primary concentration of denatured IBs and a low column of the final refolding solvent and a relatively stable refolding rate was superior to the other refolding methods. For the further purification of gene engineering Pediocin, the refolding protein was first cleft by rEK-B through which steps the thioredoxinv and the gene engineering Pediocin was separated and then enriched by the Ni-IDA agarose resin column since the His-tag of the gene engineering Pediocin could be captured by Ni in Ni-IDA agarose resin column. A 50 mmol/L iminazole was able to make the irrelevant protein eluted, and a 500 mmol/L iminazole was able to make the gene engineering pediocin eluted, and the maximize elution concentration was 0.014mg/ml.The antimicrobial activities of the purified pediocin were confermed using the indicator bacteria Lactobacillus plantarum and Listeria monocytogenes. The pediocin displayed antiseptic activities towards these two gram positive bacteria strains. It was found to be heat resistant (even maintained activity in 121℃), and kept antimicrobial activities in a broad pH extent (pH1-7). The pediocin could be degested by trypsin, pepsin, papain, proteinase K. The results indicated that the pediocin purificated by this study was proteinous.The pediocin was added to yogurt artificially contaminated with Listeria monocytogenes. The number of Listeria monocytogenes in yogurt declined gradually after the use of the pediocin.The induction methods for pediocin constructed E.coli strains and the pediocin purification technics established in this study perhaps be used in the large extent production of pediocin.