Fusion expression of pedA gene to obtain biologically active pediocin PA-1 in Escherichia coli |
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Authors: | Liu Shan-na Han Ye Zhou Zhi-jiang |
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Affiliation: | (1) College of Life Sciences, Zhejiang University, Hangzhou, 310058, China;(2) Institute of Bioengineering, Zhejiang Sci-Tech University, Hangzhou, 310018, China;(3) LC Sciences, 2575 W. Bellfort, Suite 270, Houston, TX 77054, USA;(4) Department of Biology and Biochemistry, University of Houston, Houston, TX 77004, USA; |
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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. |
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Keywords: | Bacteriocin Fusion expression Inclusion body Pediocin PA-1 Thioredoxin |
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