Similar articles

Abstract: Two heterologous expression systems using thioredoxin (trxA) as a gene fusion part in Escherichia coli were developed to produce recombinant pediocin PA-1. pediocin PA-1 structural gene pedA was isolated from Pediococcus acidilactici PA003 by the method of polymerase chain reaction (PCR), then cloned into vector pET32a(+), and expressed as thioredoxin-PedA fusion protein in the host strain E. coli BL21 (DE3). The fusion protein was in the form of inclusion body and was refolded before purification by nickel-iminodiacetic acid (Ni-IDA) agarose resin column. Biological activity of recombinant pediocin PA-1 was analyzed after cleavage of the fusion protein by enterokinase. Agar diffusion test revealed that 512-arbitrary unit (AU) recombinant pediocin PA-1 was obtained from 1 ml culture medium of E. coli (pPA003PED1) using Listeria monocytogenes as the indicator strain. thioredoxin-PedA fusion gene was further cloned into pET20b(+). thioredoxin-PedA fusion protein was detected in both the periplasmic and cytoplasmic spaces. The recombinant pediocin PA-1 from the soluble fraction attained 384 AU from 1 ml culture medium of E. coli (pPA003PED2). Therefore, biologically active pediocin PA-1 could be obtained by these two hybrid gene expression methods.

[1]Austin, C., 2003. Novel approach to obtain biologically active recombinant heterodimeric proteins in Escherichia coli. J. Chromatogr. B, 786(1-2):93-107.

[2]Beaulieu, L., Tolkatchev, D., Jetté, J.F., 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(11):1246-1258.

[3]Choi, J.H., Lee, S.Y., 2004. Secretory and extracellular production of recombinant proteins using Escherichia coli. Appl. Microbiol. Biotechnol., 64(5):625-635.

[4]Fu, X.Y., Tong, W.Y., Wei, D.Z., 2005. Extracellular production of human parathyroid hormone as a thioredoxin fusion form in Escherichia coli by chemical permeabilization combined with heat treatment. Biotechnol. Prog., 21(5):1429-1435.

[5]Gillor, O., Etzion, A., Riley, M.A., 2008. The dual role of bacteriocins as anti- and probiotics. Appl. Microbiol. Biotechnol., 81(4):591-606.

[6]Ingham, A.B., Moore, R.J., 2007. Recombinant production of antimicrobial peptides in heterologous microbial systems. Biotechnol. Appl. Biochem., 47(1):1-9.

[7]Kheadr, E., Zihler, A., Dabour, N., Lacroix, C., Blay, G.L., Fliss, I., 2010. Study of the physicochemical and biological stability of pediocin PA-1 in the upper gastrointestinal tract conditions using a dynamic in vitro model. J. Appl. Microbiol., 109(1):54-64.

[8]Lee, J.H., Kim, J.H., Hwang, S.W., Lee, W.J., Yoon, H.K., Lee, H.S., Hong, S.S., 2000. High-level expression of antimicrobial peptide mediated by a fusion partner reinforcing formation of inclusion bodies. Biochem. Biophys. Res. Commun., 277(3):575-580.

[9]Makrides, S.C., 1996. Strategies for achieving high-level expression of genes in Escherichia coli. Microbiol. Rev., 60(3):512-538.

[10]Sambrook, J., Russell, D.W., 2001. Molecular Cloning: A Laboratory Manual, 3rd Ed. Cold Spring Harbor Laboratory Press, New York, p.1-170.

[11]Sommer, B., Friehs, K., Flaschel, E., 2010. Efficient production of extracellular proteins with Escherichia coli by means of optimized coexpression of bacteriocin release proteins. J. Biotechnol., 145(4):350-358.

[12]Tian, Z.G., Teng, D., Yang, Y.L., Luo, J., Feng, X.J., Fan, Y., Zhang, F., Wang, J.H., 2007. Multimerization and fusion expression of bovine lactoferricin derivative LfcinB15-W4,10 in Escherichia coli. Appl. Microbiol. Biotechnol., 75(1):117-124.

[13]Yildirim, S., Konrad, D., Calvez, S., Drider, D., Prevost, H., Lacroix, C., 2007. Production of recombinant bacteriocin divercin V41 by high cell density Escherichia coli batch and fed-batch cultures. Appl. Microbiol. Biotechnol., 77(3):525-531.

[14]Zhou, Z.J., Han, Y., Han, X., Zheng, F., 2006. Isolation of bacteriocin-producing Pediococcus acidilactici strain from fermented Chinese cabbage. Food Sci., 27(4):89-92 (in Chinese).