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Cloning and heterologous expression of plnE, -F, -J and -K genes derived from soil metagenome and purification of active plantaricin peptides

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Abstract

Plantaricin gene-specific primers were used to obtain plnE, -F, -J and -K structural gene amplicons from soil metagenome. These amplicons were cloned and expressed in pET32a (+) vector in Escherichia coli BL21 (DE3). PlnE, -F, -J and -K peptides were expressed as His-tagged-fusion proteins and were separated by Ni2+ -chelating affinity chromatography. The peptides were released from the fusion by enterokinase cleavage and separated from the carrier thioredoxin. The cleaved peptides were further analysed for antimicrobial activity and found to be active against Listeria innocua NRRL B33314, Micrococcus luteus MTCC 106 and lactic acid bacteria, such as Enterococcus casseliflavus NRRL B3502, Lactococcus lactis lactis NRRL 1821, Lactobacillus curvatus NRRL B4562 and Lactobacillus plantarum NRRL B4496. E. coli has been successfully exploited as a host for heterologous expression with a significant yield of fused and cleaved peptides in the range of 8–12 and 1–1.5 mg/l of the culture, respectively. Heterologous expression, therefore, can be used to overcome the constraints of low yield often reported from a native strain.

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Acknowledgments

The authors acknowledge financial assistance and the facilities supported by University Grant Commission (SAP) and Department of Science and Technology (FIST), Govt. of India in Department of Genetics, UDSC. SS acknowledges the financial assistance from Department of Biotechnology, and GP acknowledges Council of Scientific and Industrial Research, Government of India for providing fellowship.

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  1. Department of Genetics, University of Delhi South Campus, New Delhi, 110021, India

    Gargi Pal & Sheela Srivastava

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  1. Gargi Pal
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  2. Sheela Srivastava
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Correspondence to Sheela Srivastava.

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Pal, G., Srivastava, S. Cloning and heterologous expression of plnE, -F, -J and -K genes derived from soil metagenome and purification of active plantaricin peptides. Appl Microbiol Biotechnol 98, 1441–1447 (2014). https://doi.org/10.1007/s00253-013-5097-1

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  • DOI: https://doi.org/10.1007/s00253-013-5097-1

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