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Investigating a putative transcriptional regulatory protein encoded by Rv1719 gene of Mycobacterium tuberculosis

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Abstract

Mycobacterium tuberculosis, the causative agent of tuberculosis, demonstrates immense plasticity with which it adapts to a highly dynamic and hostile host environment. This is facilitated by a web of signalling pathways constantly modulated by a multitude of proteins that regulate the flow of genetic information inside the pathogen. Transcription factors (TFs) belongs to one such family of proteins that modulate the signalling by regulating the abundance of proteins at the transcript level. In the current study, we have characterized the putative transcriptional regulatory protein encoded by the Rv1719 gene of Mycobacterium tuberculosis. This TF belongs to the IclR family of proteins with orthologs found in both bacterial and archaeal species. We cloned the Rv1719 gene into the pET28a expression vector and performed heterologous expression of the recombinant protein with E coli as the host. Further, optimization of the purification protocol by affinity chromatography and characterization of proteins for their functional viability has been demonstrated using various biochemical and/or biophysical approaches. Scale-up of purification yielded approximately 30 mg of ~ 28 kDa protein per litre of culture. In-silico protein domain analysis of Rv1719 protein predicted the presence of the helix-turn-helix (HTH) domain suggesting its ability to bind DNA sequence and modulate transcription; a hallmark of a transcriptional regulatory protein. Further, by performing electrophoretic mobility shift assay (EMSA) we demonstrated that the protein binds to a specific DNA fragment harboring the probable binding site of one of the predicted promoters.

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Acknowledgements

This study was supported by Indian Council of Medical Research (Project ID; TB-Fellowship/29/2018-ECD-1) to MP, India-Singapore grant jointly sponsored by the Department of Science and Technology, India, and by A*STAR, Singapore, to A.K.P. (Project ID; INT/Sin/P-08/2015) and Intramural funding from Translational Health Science and Technology Institute (THSTI) to AKP. The funder of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication.

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Authors and Affiliations

  1. Mycobacterial Pathogenesis Laboratory, Translational Health Science and Technology Institute, Faridabad, Haryana, India

    Manitosh Pandey, Chandresh Sharma & Amit Kumar Pandey

  2. Department of Life Science, ITM University, Gwalior, Madhya Pradesh, India

    Manitosh Pandey & Sonia Johri

  3. Department of Life Sciences and Biotechnology, Chhatrapati Shahu Ji Maharaj University, Kanpur, Uttar Pradesh, India

    Chandresh Sharma

  4. National Institute of Immunology, New Delhi, India

    Satish Tiwari & Bichitra K Biswal

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  1. Manitosh Pandey
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  2. Satish Tiwari
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  4. Bichitra K Biswal
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Contributions

AKP is the principal investigator of the study. AKP and CS contributed towards the conception and design of the study. MP, CS were responsible for the acquisition of data. ST performed the GFC analysis. MP, CS and AKP analysed and interpreted the data, with significant inputs from ST, SJ, BKB. MP, CS, AKP drafted the manuscript and all authors were involved in revising it critically for intellectual content, and have given final approval of the version to be published.

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Correspondence to Chandresh Sharma or Amit Kumar Pandey.

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Pandey, M., Tiwari, S., Johri, S. et al. Investigating a putative transcriptional regulatory protein encoded by Rv1719 gene of Mycobacterium tuberculosis. Protein J 41, 424–433 (2022). https://doi.org/10.1007/s10930-022-10062-9

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