title: Cloning and heterologous expression of plnE, -F, -J and -K genes derived from soil metagenome and purification of active plantaricin peptides.
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 Ni(2+) -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.
authors.affiliation: Department of Genetics, University of Delhi South Campus, New Delhi, 110021, India.
authors.forename: Gargi
authors.initials: G
authors.lastname: Pal
authors.forename: Sheela
authors.initials: S
authors.lastname: Srivastava
mesh_terms: D000900:Anti-Bacterial Agents / Q000302:isolation & purification / Q000494:pharmacology*; D001430:Bacteriocins / Q000235:genetics / Q000302:isolation & purification* / Q000494:pharmacology*; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics* / Q000302:isolation & purification; D015870:Gene Expression; D006094:Gram-Positive Bacteria / Q000187:drug effects*; D020128:Inhibitory Concentration 50; D054892:Metagenome; D008826:Microbial Sensitivity Tests; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D012988:Soil Microbiology
chemical_list: D000900:Anti-Bacterial Agents; D001430:Bacteriocins; D004269:DNA, Bacterial; D011994:Recombinant Proteins