将乳酸片球菌接种于MRS培养基,37℃静置培养17h后产生抑菌物质细菌素。用三步法从培养液中分离纯化细菌素:首先根据细菌素在pH6.0时与菌体的吸附力最强,pH2.0时吸附力最弱,通过调节pH和离心来收集细菌素,然后经过SP Sepharose Fast Flow阳离子交换柱层析和Sephadex G-50凝胶层析进行纯化,最后细菌素的得率为0.19%,比活为5.17×104AU/mg,纯化了34.54倍。得到的细菌素样品通过Tricine-SDS-PAGE电泳出现清晰条带,大小约为4600D。
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.
