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Soluble Expression and Purification of Biologically Active Human NANOG from Escherichia coli

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Healthcare Research and Related Technologies (NERC 2022)

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Abstract

NANOG is important for the early stages of embryogenesis and embryonic stem cell maintenance. It is also involved in the nuclear reprogramming necessary for forming induced pluripotent stem cells, thus offering enormous potential for cell therapies. Here, the expression and purification of human NANOG protein from the soluble fraction of bacterial cell lysate are reported. To produce fusion tagged transducible version of NANOG protein, we fused the human NANOG cDNA sequence to different fusion tags for affinity purification and efficient cell and nuclear delivery. Subsequently, the optimal expression conditions were identified, and the recombinant fusion protein was purified to homogeneity with a retained secondary structure. Additionally, NANOG is linked to cellular multipotency, which plays crucial roles in stemness, tumor progression and migration, thus highlighting its potential role as a therapeutic target for a myriad of malignancies such as cervical cancer. Therefore, we investigated the effect of application of the HTN-NANOG fusion protein on HeLa cells. Upon protein transduction in HeLa cells, the cell migration rate was augmented in the presence of NANOG. Furthermore, we observed the downregulation of p27 in HeLa cells in the presence of NANOG at mRNA and protein levels. From a future perspective, this bioactive form of human NANOG protein can be used to understand the molecular roles of NANOG in several cellular processes associated with cancer and induced pluripotent stem cells.

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Acknowledgements

North Eastern Region—Biotechnology Programme Management Cell (BT/PR16655/NER/95/132/2015), DBT, Govt. of India funded this study.

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Authors and Affiliations

  1. Laboratory for Stem Cell Engineering and Regenerative Medicine, Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, 781039, Assam, India

    Madhuri Thool & Rajkumar P. Thummer

  2. Department of Biotechnology, National Institute of Pharmaceutical Education and Research Guwahati, Changsari, Guwahati, 781101, Assam, India

    Madhuri Thool & S. Sudhagar

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  1. Madhuri Thool
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  2. S. Sudhagar
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  3. Rajkumar P. Thummer
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Contributions

MT designed and performed the experiments, assembled and analyzed the data, wrote the manuscript; SS analyzed and interpreted the data; and RPT conceptualized and designed the study, analyzed the data, supervised the experiments, wrote the manuscript, and provided financial support. The final draft of the manuscript was approved by all the authors for publication.

Corresponding author

Correspondence to Rajkumar P. Thummer .

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Editors and Affiliations

  1. Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, Assam, India

    Lalit M. Pandey

  2. Department of Chemical Engineering, Indian Institute of Technology Guwahati, Guwahati, Assam, India

    Raghvendra Gupta

  3. Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, Assam, India

    Rajkumar P. Thummer

  4. Jyoti and Bhupat Mehta School of Health Sciences and Technology, Indian Institute of Technology Guwahati, Guwahati, Assam, India

    Rajiv Kumar Kar

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Conclusion

The purification of HTN-NANOG fusion protein from E. coli with a retained secondary structure and its bioactivity in human cells is demonstrated in this study. This study reported that human NANOG fusion protein enhanced proliferation, migration and downregulated p27 gene expression in HeLa cells, demonstrating its bioactivity. This biologically active human NANOG protein can be utilized to elucidate the biological function of NANOG in various cellular processes associated with cancer and induced pluripotent stem cells.

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Thool, M., Sudhagar, S., Thummer, R.P. (2023). Soluble Expression and Purification of Biologically Active Human NANOG from Escherichia coli. In: Pandey, L.M., Gupta, R., Thummer, R.P., Kar, R.K. (eds) Healthcare Research and Related Technologies. NERC 2022. Springer, Singapore. https://doi.org/10.1007/978-981-99-4056-1_6

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