Abstract
The NB-C1 gene, acquired from the result of data mining of the lactic acid bacteria genome, is a novel potential class IIa bacteriocin gene with the characteristic YGNGVxC cluster. To produce soluble NB-C1 efficiently and overcome issues of protein toxicity, we adopted a GFP fusion strategy using an Escherichia coli cell-free protein expression system. We constructed the expression vector pIVEX2.4d-GFP-NB-C1, which was expressed in both the batch mode and the continuous exchange cell-free (CECF) systems. The amount of soluble fusion protein achieved from the CECF system (2.2 mg/ml) was approximately three times higher than that in the batch mode (0.73 mg/ml). The soluble fusion protein was purified via one-step Ni–NTA affinity chromatography, with a concentration of 0.26 mg/ml and a purity of 95%. The purified NB-C1 showed strong antimicrobial activity against the indicator bacteria Listeria monocytogenes.
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References
Barrell PJ, Liew OW, Conner AJ (2004) Expressing an antibacterial protein in bacteria for raising antibodies. Protein Expr Purif 33:153–159
Beaulieu L, Groleau D, Miguez CB, Jetté JF, Aomari H, Subirade M (2005) Production of pediocin PA-1 in the methylotrophic yeast Pichia pastoris reveals unexpected inhibition of its biological activity due to the presence of collagen-like material. Protein Expr Purif 43:l11–l125
Beaulieu L, Tolkatchev D, Jetté JF, Groleau D, Subirade M (2007) Production of active pediocin PA-1 in Escherichia coli using a thioredoxin gene fusion expression approach: cloning, expression, purification, and characterization. Can J Microbiol 53:1246–1258
Chen HQ, Xu ZN, Xu NZ, Cen PL (2005) Efficient production of a soluble fusion protein containing human beta-defensin-2 in E. coli cell-free system. J Biotechnol 115:307–315
Chen HQ, Fan LM, Xu ZN, Yin XF, Cen PL (2007) Efficient production of soluble human beta-defensin-3–4 fusion proteins in Escherichia coli cell-free system. Proc Biochem 42:423–428
Cipáková I, Hostinová E, Gasperík J, Velebný V (2004) High-level expression and purification of a recombinant hBD-1 fused to LMM protein in Escherichia coli. Protein Expr Purif 37:207–212
Drider D, Fimland G, Héchard Y, McMullen LM, Prévost H (2006) The continuing story of class IIa bacteriocins. Microbiol Mol Biol Rev 70:564–582
Georgopoulos A (1978) A simple micro agar diffusion method for the determination of antibiotic concentrations in blood and other body fluids. Zentralbl Bakteriol Orig A 242:387–393
Moon GS, Pyun YR, Kim WJ (2006) Expression and purification of a fusion-typed pediocin PA-1 in Escherichia coli and recovery of biologically active pediocin PA-1. Int J Food Microbiol 108:136–140
Morisset D, Frère J (2002) Heterologous expression of bacteriocins using the mesentericin Y105 dedicated transport system by Leuconostoc mesenteroides. Biochimie 84:569–576
Rajesh S, Knowles T, Overduin M (2011) Production of membrane proteins without cells or detergents. Nature Biotechnol 28:250–254
Richard C, Drider D, Elmorjani K, Marion D, Prévost H (2004) Heterologous expression and purification of active divercin V41, a class IIa bacteriocin encoded by a synthetic gene in Escherichia coli. J Bacteriol 186:4276–4284
Sambrook J, Russell DW (2002) Molecular cloning: a laboratory manual, 3rd edn, Beijing. Cold Spring Harbor Laboratory Press, New York
Valore EV, Ganz T (1997) Laboratory production of antimicrobial peptides in native conformation. Methods Mol Biol 78:115–131
Wuu JJ, Swartz JR (2008) High yield cell-free production of integral membrane proteins without refolding or detergents. Biochim Biophys Acta 1778:1237–1250
Xie Y, Chen HQ, Zhang QX, Tian FW, Chen YQ, Zhang H, Chen W (2011) Expression and characterization of a new class IIa bacteriocin. Chin J Biotech 27:976–982
Acknowledgments
This study was financially supported by the National Natural Science Foundation of China (No. 20706023), Research Program of State Key Laboratory of Food Science and Technology (SKLFMB-200802), Fundamental Research Funds for the Central Universities (JUSRP11017 and JUSRP31002), and Innovative Research Team in University (IRT0627). We thank Isabelle M. Berquin for editing the manuscript.
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Chen, H., Yan, X., Tian, F. et al. Cloning, expression, and identification of a novel class IIa bacteriocin in the Escherichia coli cell-free protein expression system. Biotechnol Lett 34, 359–364 (2012). https://doi.org/10.1007/s10529-011-0779-1
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DOI: https://doi.org/10.1007/s10529-011-0779-1