| The subclass classⅡa bacteriocins among the bacteriocins produced by lactic acid bacteria (LAB), are defined as antilisteria and heat-stable small antipeptides, which can be digested by enzymes and do harmless to human health. For their most potential function in controlling the contamination of the pathogenic listeria and their application in food preservation, classⅡa bacteriocins have received particular attention in these years.More than twenty kinds of classⅡa bacteriocins found these years were obtained mostly by conventional strains screening method, and needed complicated isolation and purification processes. With many interferential factors and low production yield, it is difficult to screen out new bacteriocins with conventional method. Considerding the strict conservative featrue of classⅡa bacteriocin sequences, the employments of genomics and bioinformatics for the exploration of new classⅡa bacteriocins became an effective method. And the heterogenous expression of bacteriocins had overcome the restriction of the regulation from producer strains. Therefore the continuous production and overexpression of bacteriocins came true. The primary expression systems for classⅡa bacteriocins were E.coli, LAB and yeast, of which the E.coli system was most frequently used, and the expressed bacteriocins were mostly existed as fusion inclusion bodies in cytoplasm, resulting in the complicated subsequent processes. In the recent decades, the rapidly developing cell-free protein synthesis system had successfully set up the link between the gene and the protein, and enabled the fast expression of heterogenous genes in vitro, avoiding the interferences of the complicated regulation and the metabolism of producer organisms and the toxicity of antipeptides to the expression hosts. Thus, the cell-free protein synthesis system is a potential alternative system for the recombinant expression of classⅡa bacteriocins.In this paper, a potential novel classⅡa bacteriocin gene was studied, which was mined from the genome of sequenced LAB through the instrument of genomics and bioinformatics. Different strategies of recombinant expression had been carried out, some of which achieved efficient and active production of NB-C1 fusion protein. Furthermore, the antibacterial activity of fusion NB-C1 was indentified. Firstly, the expression vector harboring NB-C1 gene were constructed including the following vectors, such as pIVEX2.4d-NB-C1, pIVEX 2.4d-GFP-NB-C1 (with green fluorescent protein (GFP) fusion tag) and pIVEX 2.4d-TrxA-NB-C1 (with thioredoxin (TrxA) fusion tag). Secondly, the recombinant plasmids were expressed in E.coli hosts (BL21(DE3) and BL21(DE3)plysS) and the E.coli cell-free system, respectively, and the fusion GFP-NB-C1 protein was successfully expressed in a soluble active form in both in vivo and in vitro system with the yield of 840μg/mL (in vivo), 730μg/mL (Batch-mode) and 2.2 mg/mL(continuous exchange cell-free, CECF mode), respectively, while the fusion TrxA-NB-C1 protein was existed as inclusion body in cellular cytoplasm and as soluble fusion protein in cell-free system with low production. The yield of GFP-NB-C1 from BL21(DE3)plysS and the CECF system after Ni-NTA affinity purification were 36 mg/L with the purity of 95% and 0.26 mg/mL with the purity of 98%, respectively. Finally, the antimicrobial activity of these recombinant proteins were indentified, the results showed that the soluble fusion NB-C1 proteins had significant inhibition effects on listeria monocytogenes.The successful recombinant expression of NB-C1 gene and the investigation of soluble production levels with different expression strategies indicated that the way from classⅡa bacteriocin genes to active proteins was constructed successfully. This study would be a reference to the mining and characterization of novel classⅡa bacteriocins.
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