Construction, heterologous expression, partial purification, and in vitro cytotoxicity of the recombinant plantaricin E produced by Lactococcus lactis against Enteropathogenic Escherichia coli K.1.1 and human cervical carcinoma (HeLa) cells.

Abstract

Lactobacillus plantarum produces bacteriocin called plantaricin that can kill or inhibit other bacteria. Plantaricin E (Pln E), a recombinant bacteriocin, has been successfully constructed and produced by a GRAS host, Lactococcus lactis. A polymerase chain reaction (PCR) overlapping technique has been used to construct a ligation of signal peptide gene, Pln A and bacteriocin encoding gene, Pln E. Furthermore, the fusion fragment were cloned into pNZ8148 vector and transformed into L. lactis NZ3900. Molecular expression study shows that recombinant L. lactis NZ3900 is able to express the mature pln E at transcription level with size of 168bp. Plantaricin E is purified by ammonium sulphate precipitation followed by gel filtration chromatography. Purified fractions were proven to be active against Enteropathogenic Escherichia coli K.1.1. The other fractions of Pln E also have antibacterial activity against several Gram positive and Gram negative bacteria. Purified recombinant plantaricin E is 3.7kDa in size. The cytotoxicity assay shows purified Pln E inhibits 46.949 ± 3.338% of HeLa cell lines on 10ppm dose whilst the metabolite inhibits 53.487 ± 2.957% of HeLa cell line on 100ppm dose. The IC50 calculation of Pln E metabolite is 107.453ppm, while the purified protein is 11.613ppm.