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Structural and functional evaluation of recombinant histidine phosphokinase NisK and response regulator NisR: in silico and experimental approach.
Heidari, Sahar; Hamedi, Javad; Olad, Gholamreza; Amani, Jafar; Rastegar Shariat Panahi, Mona; Najafi, Ali.
Afiliação
  • Heidari S; Applied Biotechnology Research Center, Baqiyatallah University of Medical Sciences, Vanak Sq. Molasadra St., P.O. Box 19395-5487, Tehran, Iran.
  • Hamedi J; Department of Microbial Biotechnology, School of Biology, College of Science, University of Tehran, Tehran, Iran.
  • Olad G; Applied Biotechnology Research Center, Baqiyatallah University of Medical Sciences, Vanak Sq. Molasadra St., P.O. Box 19395-5487, Tehran, Iran. grolad@gmail.com.
  • Amani J; Applied Microbiology Research Center, Systems Biology and Poisonings Institute, Baqiyatallah University of Medical Sciences, Vanak Sq. Molasadra St., P.O. Box 19395-5487, Tehran, Iran. jafar.amani@gmail.com.
  • Rastegar Shariat Panahi M; Department of Biochemistry, Payame Noor University, Tehran, Iran.
  • Najafi A; Molecular Biology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran.
World J Microbiol Biotechnol ; 35(11): 169, 2019 Oct 25.
Article em En | MEDLINE | ID: mdl-31654140
In the two-component system of NisRK from Lactococcus lactis, the production of nisin is affected by transmembrane NisK and activation of intracellular NisR. The transcription of nisin structural genes can be induced by derivatives of nisin. NisR activation leads to the activation of nisA/Z transcription, which encodes the nisin maturation machinery, nisin regulation and activation of the nisFEG operon to confer immunity. The aim of this study was to express the Lactococcus lactis histidine phosphokinase NisK and response regulator NisR in E. coli, and to perform activity assays and in silico analysis. In silico methods were applied to study the properties and structures of the NisK and NisR proteins, including prediction of physicochemical characteristics, secondary and tertiary structure, stability and ligand-receptor interactions.pET32a and pET28a vectors containing synthetic nisK and nisR genes were transformed into E. coli followed by IPTG induction. SDS-PAGE and western blotting methods were applied to confirm the presence and identity of the amplified proteins. Following purification, the proteins were dialyzed and then prepared for activity assay. The CAI index showed that the genes was compatible with the E. coli host and that the proteins have effective expression. Also, the mRNA prediction results suggest that there is enough mRNA stability for efficient translation in the new host. NisK and NisR recombinant proteins were expressed in E. coli with half - lives of around 10 h and were confirmed with molecular weights of 27 kDa and 69 kDa, respectively, by SDS-PAGE and western blotting. The secondary structure of the recombinant proteins as predicted by circular dichroism spectroscopy was similar to the in silico protein structures. Activity assay of recombinant NisK was performed by measuring the amount of consumed ATP according to the light produced by luciferase. Because NisK and NisR have a direct impact on each other, they have an essential role in increasing the production of nisin and they can be used in different research fields. Our results demonstrated that recombinant proteins NisK and NisR preserved their structure and function after expression.
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Texto completo: 1 Coleções: 01-internacional Contexto em Saúde: 3_ND Base de dados: MEDLINE Assunto principal: Proteínas de Bactérias / Fatores de Transcrição / Proteínas Recombinantes / Regulação Bacteriana da Expressão Gênica / Lactococcus lactis / Histidina Quinase / Genes Bacterianos Tipo de estudo: Prognostic_studies Idioma: En Revista: World J Microbiol Biotechnol Ano de publicação: 2019 Tipo de documento: Article

Texto completo: 1 Coleções: 01-internacional Contexto em Saúde: 3_ND Base de dados: MEDLINE Assunto principal: Proteínas de Bactérias / Fatores de Transcrição / Proteínas Recombinantes / Regulação Bacteriana da Expressão Gênica / Lactococcus lactis / Histidina Quinase / Genes Bacterianos Tipo de estudo: Prognostic_studies Idioma: En Revista: World J Microbiol Biotechnol Ano de publicação: 2019 Tipo de documento: Article