title: Improved expression and purification of the correctly folded response regulator PlnC from lactobacilli.
abstract: The response regulator PlnC is part of the signal transduction system that plays a key role in the regulation of bacteriocin production in Lactobacillus plantarum C11. In this study, we wanted to express high levels of the response regulator PlnC in a soluble and native form for purification and further studies. The protein was expressed as a fusion protein (fPlnC) containing an N-terminal Flag-tag to facilitate detection and purification. When the fusion gene, fplnC, was expressed in Escherichia coli BL21, nearly all (99%) of the recombinant protein ended up inside inclusion bodies as an incorrectly folded protein. By utilizing two different Gram-positive expression systems (SIP and NICE) in L. plantarum NC8 and Lactobacillus sakei Lb790, the expression of the soluble fPlnC was significantly increased, being 20-40 times more than that in E. coli BL21. Using the N-terminal tag, the expressed protein was purified by immunoprecipitation. By DNA-binding study (EMSA), we demonstrated that the fusion protein purified from the soluble pool was correctly folded as judged by its ability to bind specifically on regulated promoters. Using our approach, we estimate that about 1 mg of fPlnC can be purified from 11 of the bacterial culture.
authors.affiliation: Laboratory for Microbial Gene Technology, Department of Biotechnological Sciences, The Norwegian University of Life Science, PO Box 5003, N-1432 As, Norway.
authors.forename: Daniel
authors.initials: D
authors.lastname: Straume
authors.forename: Lars
authors.initials: L
authors.lastname: Axelsson
authors.forename: Ingolf F
authors.initials: IF
authors.lastname: Nes
authors.forename: Dzung B
authors.initials: DB
authors.lastname: Diep
mesh_terms: D001430:Bacteriocins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D015153:Blotting, Western; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D048191:Lactiplantibacillus plantarum / Q000235:genetics*; D009842:Oligopeptides; D010455:Peptides / Q000737:chemistry / Q000235:genetics; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics
chemical_list: D001430:Bacteriocins; D004271:DNA, Fungal; D009842:Oligopeptides; D010455:Peptides; D011994:Recombinant Proteins; C085706:FLAG peptide