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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

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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 168 bp. 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 3.7 kDa in size. The cytotoxicity assay shows purified Pln E inhibits 46.949 ± 3.338% of HeLa cell lines on 10 ppm dose whilst the metabolite inhibits 53.487 ± 2.957% of HeLa cell line on 100 ppm dose. The IC50 calculation of Pln E metabolite is 107.453 ppm, while the purified protein is 11.613 ppm.

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Acknowledgements

This research was funded by Insentif Riset Sistem Inovasi Nasional (INSINAS) program from Ministry of Research, Technology, and Higher Education fiscal year 2016–2017 and KKP3N Program from Indonesian Agency for Agricultural Research and Development (IAARD), Ministry of Agriculture fiscal year 2014–2015. All facilities were supported by Biotechnology-Indonesian Institute of Science (LIPI) and Indonesian Center for Agricultural Biotechnology and Genetic Resources Research and Development (ICABIOGRAD), Ministry of Agriculture. The authors would like to thank to Muhminah for her assistance.

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Authors and Affiliations

  1. Research Center for Biotechnology, Indonesia Institute of Science (LIPI), Raya Bogor Street Km. 46, Cibinong, Bogor, West Java, 16911, Indonesia

    Apon Zaenal Mustopa, Hidayah Murtiyaningsih & Wida Nurul Alfisyahrin

  2. School of Biotechnology, Bogor Agricultural University, Bogor, Indonesia

    Siti Mariyah & Sri Budiarti

  3. Indonesian Center for Agricultural Biotechnology and Genetic Resources Research and Development (ICABIOGRAD), Cimanggu, Bogor, Indonesia

    Fatimah

  4. Research Center for Bioresources and Biotechnology, Bogor Agricultural University, Bogor, Indonesia

    Sri Budiarti

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  1. Apon Zaenal Mustopa
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Correspondence to Apon Zaenal Mustopa.

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Mustopa, A.Z., Mariyah, S., Fatimah et al. 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. Mol Biol Rep 45, 1235–1244 (2018). https://doi.org/10.1007/s11033-018-4277-6

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