title: Fusion expression of pedA gene to obtain biologically active pediocin PA-1 in Escherichia coli.
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.
authors.affiliation: School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China.
authors.forename: Shan-na
authors.initials: SN
authors.lastname: Liu
authors.forename: Ye
authors.initials: Y
authors.lastname: Han
authors.forename: Zhi-jiang
authors.initials: ZJ
authors.lastname: Zhou
mesh_terms: D001430:Bacteriocins / Q000096:biosynthesis* / Q000235:genetics* / Q000494:pharmacology; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004765:Enteropeptidase; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000096:biosynthesis / Q000235:genetics; D015870:Gene Expression; D050939:Gene Fusion; D005798:Genes, Bacterial; D002479:Inclusion Bodies / Q000378:metabolism; D008089:Listeria monocytogenes / Q000187:drug effects; D000070017:Pediocins; D010376:Pediococcus / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology; D013879:Thioredoxins / Q000096:biosynthesis / Q000235:genetics
chemical_list: D001430:Bacteriocins; D017931:DNA Primers; D029968:Escherichia coli Proteins; D000070017:Pediocins; D011993:Recombinant Fusion Proteins; C517609:TrxA protein, E coli; C074288:pediocin PA-1; D013879:Thioredoxins; D004765:Enteropeptidase