Codon Optimization, Cloning, Expression, Purification, and Secondary Structure Determination of Human ETS2 Transcription
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ORIGINAL PAPER
Codon Optimization, Cloning, Expression, Purification, and Secondary Structure Determination of Human ETS2 Transcription Factor Krishna Kumar Haridhasapavalan1 · Pradeep Kumar Sundaravadivelu1 · Rajkumar P. Thummer1 Accepted: 7 August 2020 © Springer Science+Business Media, LLC, part of Springer Nature 2020
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. Keywords ETS2 · E. coli · Protein expression · Protein purification · Recombinant protein · Secondary structure
Introduction Recombinant protein production has transformed the field of biotechnology, and to date, numerous recombinant proteins have been commercially used for various biomedical Electronic supplementary material The online version of this article (https://doi.org/10.1007/s12033-020-00266-8) contains supplementary material, which is available to authorized users. Krishna Kumar Haridhasapavalan and Pradeep Kumar Sundaravadivelu contributed equally to this work. * Rajkumar P. Thummer [email protected] Krishna Kumar Haridhasapavalan [email protected] Pradeep Kumar Sundaravadivelu [email protected] 1
Laboratory for Stem Cell Engineering and Regenerative Medicine, Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, Assam 781039, India
and research applications [1, 2]. Importantly, recombinant protein-mediated cellular reprogramming does not modify the genome, and therefore, it is considered to be one of the safest approaches to derive integration-free cells for biomedical applications [3, 4]. Recombinant protein also offers complete control over its time and dosage of application to determine the stage-specific role of transcription factors in the cell repr
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