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Structural and functional evaluation of recombinant histidine phosphokinase NisK and response regulator NisR: in silico and experimental approach

  • Sahar Heidari
  • Javad Hamedi
  • Gholamreza OladEmail author
  • Jafar AmaniEmail author
  • Mona Rastegar Shariat Panahi
  • Ali Najafi
Original Paper
  • 78 Downloads

Abstract

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.

Keywords

E. coli Histidine phosphokinase NisK Nisin Recombinant protein Response regulator NisR 

Notes

Acknowledgments

The authors are grateful to Dr. A.M Latifi and Dr. S. Hossein Khani for their support of this research. This study was supported by the Baqiyatallah University of Medical Sciences and Applied Biotechnology Research Center.

Funding

No funding supporter.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Informed consent

N/A.

Research involving human participants and/or animals

N/A.

Supplementary material

11274_2019_2735_MOESM1_ESM.docx (404 kb)
Supplementary file1 (DOCX 404 kb)

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Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.Applied Biotechnology Research CenterBaqiyatallah University of Medical SciencesTehranIran
  2. 2.Department of Microbial Biotechnology, School of Biology, College of ScienceUniversity of TehranTehranIran
  3. 3.Applied Microbiology Research Center, Systems Biology and Poisonings InstituteBaqiyatallah University of Medical SciencesTehranIran
  4. 4.Department of BiochemistryPayame Noor UniversityTehranIran
  5. 5.Molecular Biology Research CenterBaqiyatallah University of Medical SciencesTehranIran

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