Improved expression and purification of the correctly folded response regulator PlnC from lactobacilli.


: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.


J Microbiol Methods


Straume D,Axelsson L,Nes IF,Diep DB




Has Abstract


2006-11-01 00:00:00














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    journal_title:Journal of microbiological methods

    pub_type: 杂志文章


    authors: Nicolaisen M,Suproniene S,Nielsen LK,Lazzaro I,Spliid NH,Justesen AF

    更新日期:2009-03-01 00:00:00

  • A new recombinant protein expression system for high-throughput screening in the yeast Yarrowia lipolytica.

    abstract::Development of a high-throughput eukaryotic screening procedure is important to increase success in obtaining improved enzymes through directed enzyme evolution. This procedure was developed for the yeast Yarrowia lipolytica which becomes the second eukaryotic host for this purpose. The extracellular lipase Lip2 was u...

    journal_title:Journal of microbiological methods

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    authors: Bordes F,Fudalej F,Dossat V,Nicaud JM,Marty A

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  • Investigations of the relationship between use of in vitro cell culture-quantitative PCR and a mouse-based bioassay for evaluating critical factors affecting the disinfection performance of pulsed UV light for treating Cryptosporidium parvum oocysts in sa

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    journal_title:Journal of microbiological methods

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    authors: Garvey M,Farrell H,Cormican M,Rowan N

    更新日期:2010-03-01 00:00:00

  • Assessing the performance of novel software Strain Solution on automated discrimination of Escherichia coli serotypes and their mixtures using matrix-assisted laser desorption ionization-time of flight mass spectrometry.

    abstract::O157, O26, and O111 are the most important O serogroups of enterohemorrhagic Escherichia coli worldwide. Recently we reported a strategy for discriminating these serotypes from the others using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) based on the S10-spc-alpha operon...

    journal_title:Journal of microbiological methods

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    authors: Ojima-Kato T,Yamamoto N,Iijima Y,Tamura H

    更新日期:2015-12-01 00:00:00

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    abstract::Methods to assess the diversity of the diazotroph assemblage in the rhizosphere of the salt marsh cordgrass, Spartina alterniflora were examined. The effectiveness of nifH PCR-denaturing gradient gel electrophoresis (DGGE) was compared to that of nifH clone library analysis. Seventeen DGGE gel bands were sequenced and...

    journal_title:Journal of microbiological methods

    pub_type: 杂志文章


    authors: Lovell CR,Decker PV,Bagwell CE,Thompson S,Matsui GY

    更新日期:2008-05-01 00:00:00

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    abstract::A simple and reliable method to estimate paper degradation by cellulolytic bacteria is described. This method is based on the detection in the culture medium of a fluorescent whitening agent (FWA) added to white paper during the manufacturing process. Preliminary results using a Cellulomonas strain cultivated in a liq...

    journal_title:Journal of microbiological methods

    pub_type: 杂志文章


    authors: Bichet-Hébé I,Pourcher AM,Sutra L,Comel C,Moguedet G

    更新日期:1999-08-01 00:00:00

  • Efficient recovery of low endotoxin medium-chain-length poly([R]-3-hydroxyalkanoate) from bacterial biomass.

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    journal_title:Journal of microbiological methods

    pub_type: 杂志文章


    authors: Furrer P,Panke S,Zinn M

    更新日期:2007-04-01 00:00:00

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    journal_title:Journal of microbiological methods

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    authors: Brewster JD

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    authors: Santana MA,Moccia-V CC,Gillis AE

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    journal_title:Journal of microbiological methods

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  • Epidemiological investigation and glycotyping of clinical Pseudomonas aeruginosa isolates from patients with cystic fibrosis by mass spectrometry: association with multiple drug resistance.

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    authors: Altman E,Wang Z,Aaron SD,Liu X,Vandemheen KL,Ferris W,Giesbrecht T,Li J

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  • Improved pH-ratiometry for the three-dimensional mapping of pH microenvironments in biofilms under flow conditions.

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  • Use of Enterococcus faecalis and Bacillus atrophaeus as surrogates to establish and maintain laboratory proficiency for concentration of water samples using ultrafiltration.

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    authors: Mapp L,Klonicki P,Takundwa P,Hill VR,Schneeberger C,Knee J,Raynor M,Hwang N,Chambers Y,Miller K,Pope M

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    authors: Yang X,Beyenal H,Harkin G,Lewandowski Z

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  • Use of the frc gene as a molecular marker to characterize oxalate-oxidizing bacterial abundance and diversity structure in soil.

    abstract::Oxalate catabolism, which can have both medical and environmental implications, is performed by phylogenetically diverse bacteria. The formyl-CoA-transferase gene was chosen as a molecular marker of the oxalotrophic function. Degenerated primers were deduced from an alignment of frc gene sequences available in databas...

    journal_title:Journal of microbiological methods

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    authors: Khammar N,Martin G,Ferro K,Job D,Aragno M,Verrecchia E

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  • Simplified amplified-fragment length polymorphism method for genotyping Mycobacterium tuberculosis isolates.

    abstract::A simplified amplified-fragment length polymorphism (AFLP) method was developed and applied to genotype 52 Mycobacterium tuberculosis isolates. This method can be carried out using only one restriction enzyme (XhoI), one double strand adapter, and one PCR primer. The amounts of DNA and DNA polymerase, and the concentr...

    journal_title:Journal of microbiological methods

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    authors: Viader-Salvadó JM,Flores-Gracia J,Vega-Alonso AS,Treviño-Alvarado VM,Molina-Torres CA,Vera-Cabrera L,Guerrero-Olazarán M

    更新日期:2009-09-01 00:00:00

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    authors: Jupraputtasri W,Boonapatcharoen N,Cheevadhanarak S,Chaiprasert P,Tanticharoen M,Techkarnjanaruk S

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  • Arsenic interception by cell wall of bacteria observed with surface-enhanced Raman scattering.

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    authors: Tian H,Zhuang G,Ma A,Jing C

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  • Status of methods for assessing bacterial cell surface charge properties based on zeta potential measurements.

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    journal_title:Journal of microbiological methods

    pub_type: 杂志文章,评审


    authors: Wilson WW,Wade MM,Holman SC,Champlin FR

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  • Development of a novel plasmid vector pTIO-1 adapted for electrotransformation of Porphyromonas gingivalis.

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    journal_title:Journal of microbiological methods

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    authors: Tagawa J,Inoue T,Naito M,Sato K,Kuwahara T,Nakayama M,Nakayama K,Yamashiro T,Ohara N

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    journal_title:Journal of microbiological methods

    pub_type: 杂志文章


    authors: Ikeda M,Yamaguchi N,Tani K,Nasu M

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  • Methodological issues in the quantification of subgingival microorganisms using the checkerboard technique.

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    journal_title:Journal of microbiological methods

    pub_type: 杂志文章


    authors: Dahlen G,Preus HR,Baelum V

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    pub_type: 杂志文章


    authors: Roux-Michollet DD,Schimel JP,Holden PA

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    journal_title:Journal of microbiological methods

    pub_type: 杂志文章


    authors: Xiao D,Yang Y,Jiang W,Zhang H,Liu H,Yu H,Xie C,Zhong M,Chen L,Huang W

    更新日期:2014-10-01 00:00:00

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    journal_title:Journal of microbiological methods

    pub_type: 杂志文章


    authors: Hyeon JY,Chon JW,Choi IS,Park C,Kim DE,Seo KH

    更新日期:2012-04-01 00:00:00

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    journal_title:Journal of microbiological methods

    pub_type: 杂志文章


    authors: Huhtamella S,Leinonen M,Nieminen T,Fahnert B,Myllykoski L,Breitenstein A,Neubauer P

    更新日期:2007-03-01 00:00:00

  • Development of an oriC vector for use in Mycoplasma synoviae.

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    journal_title:Journal of microbiological methods

    pub_type: 杂志文章


    authors: Shahid MA,Marenda MS,Markham PF,Noormohammadi AH

    更新日期:2014-08-01 00:00:00