摘要
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.
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.
基金
Project supported by the National Natural Science Foundation of China (No.30571378)
the Tianjin Natural Science Foundation (No.08JCZDJC22500),China
