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Codon Optimization, Cloning, Expression, Purification, and Secondary Structure Determination of Human ETS2 Transcription Factor

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Abstract

Transcription factor ETS2 regulates genes involved in development, differentiation, angiogenesis, proliferation, and apoptosis. In addition, it is one of the core reprogramming factors responsible for the generation of human cardiomyocytes from adult somatic cells. In this study, we report the heterologous expression of human ETS2 in E. coli to produce a highly pure recombinant protein. To accomplish this, the codon-optimized 1507 bp coding sequence of the human ETS2 gene in fusion with a His-tag, a cell-penetrating peptide, and a nuclear localization sequence was cloned in the protein expression vector and transformed into E. coli strain BL21(DE3) for expression. The recombinant protein was purified to homogeneity under native conditions using immobilized metal ion affinity chromatography, and its identity was confirmed by Western blotting with an ETS2 antibody. Using far-UV circular dichroism spectroscopy, we have demonstrated that the recombinant protein has retained its secondary structure, predominantly comprising of random coils and β-sheets. Prospectively, this biological recombinant ETS2 protein can substitute viral and genetic forms of ETS2 in a cell reprogramming process to facilitate the generation of clinical-grade cells. It can also be used to investigate its molecular role in various biological processes and diseases and for biochemical and structural studies.

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Acknowledgements

We thank all the members of the Laboratory for Stem Cell Engineering and Regenerative Medicine (SCERM) for their critical reading and excellent support. This work was supported by a research grant from Science and Engineering Research Board (SERB), Department of Science and Technology, Government of India (Early Career Research Award; ECR/2015/000193).

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  1. Krishna Kumar Haridhasapavalan and Pradeep Kumar Sundaravadivelu contributed equally to this work.

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

    Krishna Kumar Haridhasapavalan, Pradeep Kumar Sundaravadivelu & Rajkumar P. Thummer

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  1. Krishna Kumar Haridhasapavalan
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KKH was responsible for conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing, and final approval of the manuscript; PKS was responsible for collection and/or assembly of data, data analysis and interpretation, and final editing and approval of the manuscript; RPT was responsible for conception and design, data analysis and interpretation, manuscript writing, final approval of manuscript, and financial support. All the authors gave consent for publication.

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Correspondence to Rajkumar P. Thummer.

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Haridhasapavalan, K.K., Sundaravadivelu, P.K. & Thummer, R.P. Codon Optimization, Cloning, Expression, Purification, and Secondary Structure Determination of Human ETS2 Transcription Factor. Mol Biotechnol 62, 485–494 (2020). https://doi.org/10.1007/s12033-020-00266-8

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