关键词: 细菌素, 乳酸片球菌素, 原核表达, 蛋白纯化

Abstract:

Pediocin PA-1 is a representative class IIa bacteriocin produced by Pediococci with the potential to serve as a
food-grade preservative for controlling Listeria contamination. To obtain its soluble expression, Pediocin PA-1 structural
and immunity gene pedAB was amplified by polymerase chain reaction (PCR) from Pediococcus acidilactici PAF, and
cloned into the vector pGEX-6p-1 to construct the pGEX/his-pedAB encoding the recombinant pediocin PA-1 with
GST-His-DDDDK in the N-terminal of pediocin PA-1. The plasmid pGEX/his-pedAB was then transformed into
Escherichia coli Rosetta (DE3). After induction with isopropyl-β-D-thiogalactopyranoside, the recombinant pediocin
PA-1 was successfully expressed in E. coli cytoplasm. The expressed fusion protein was purified by Ni-NTA metal affinity
chromatography, subsequently loaded to GST affinity column and treated with recombinant bovine enterokinase. Mature
pediocin PA-1 was liberated from the recombinant protein. The purity of pediocin PA-1 was detected by high-performance
liquid chromatography and mass spectrometry. The antibacterial activity of pediocin PA-1 was determined by agar diffusion
method using Listeria monocytogenes CMCC54004 as an indicator. The results showed that fusion pediocin PA-1 did not
have bactericidal activity against Listeria monocytogenes, but when the GST-His-DDDDK in the N-terminal was removed,
the cleaved pediocin PA-1 restored its bactericidal activity. The purified pediocin PA-1 had a purity above 90%.

Key words: bacteriocin, pediocin, prokaryotic expression, protein purification