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Generation of a Recombinant Stem Cell-Specific Human SOX2 Protein from Escherichia coli Under Native Conditions

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Abstract

The stem cell-specific SOX2 transcription factor is critical for early embryonic development and the maintenance of embryonic and neural stem cell identity. It is also crucial for the generation of induced pluripotent and neural stem cells, thus providing immense prospect in patient-specific therapies. Here, we report soluble expression and purification of human SOX2 protein under native conditions from a bacterial system. To generate this macromolecule, we codon-optimized the protein-coding sequence and fused it to a nuclear localization signal, a protein transduction domain, and a His-tag. This was then cloned into a protein expression vector and was expressed in Escherichia coli. Subsequently, we have screened and identified the optimal expression conditions to obtain recombinant fusion protein in a soluble form and studied its expression concerning the position of fusion tags at either terminal. Furthermore, we purified two versions of recombinant SOX2 fusion proteins to homogeneity under native conditions and demonstrated that they maintained their secondary structure. This molecular tool can substitute genetic and viral forms of SOX2 to facilitate the derivation of integration-free induced pluripotent and neural stem cells. Furthermore, it can be used in elucidating its role in stem cells, various cellular processes and diseases, and for structural and biochemical studies.

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Acknowledgments

We thank all the members of the Laboratory for Stem Cell Engineering and Regenerative Medicine (SCERM) for their critical reading and excellent support. The authors gratefully acknowledge the support of DBT Program Support (Prof. S.S. Ghosh), Department of Biosciences and Bioengineering, IIT Guwahati, for their assistance in Circular Dichroism experiments. This work was supported by North Eastern Region—Biotechnology Programme Management Cell (NERBPMC), Department of Biotechnology, Government of India (BT/PR16655/NER/95/132/2015), and also by IIT Guwahati Institutional Top-Up on Start-Up Grant.

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  1. Madhuri Thool and Chandrima Dey have been contributed equally to this work.

Authors and Affiliations

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

    Madhuri Thool, Chandrima Dey & Rajkumar P. Thummer

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

    Madhuri Thool & S. Sudhagar

  3. Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, Assam, 781039, India

    Srirupa Bhattacharyya

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  1. Madhuri Thool
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MT and CD were responsible for conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing, and final approval of the manuscript; SB was responsible for collection and/or assembly of data, data analysis and interpretation, and final editing and approval of the manuscript; SS was responsible for conception and design, data analysis and interpretation, final approval of manuscript and financial support; and RPT was responsible for conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing, final approval of manuscript and financial support.

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Correspondence to Rajkumar P. Thummer.

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12033_2021_305_MOESM1_ESM.tif

Supplementary file1 (TIF 14273 KB) Fig. S1 Evaluation of human SOX2 protein-coding sequence before (non-optimized) and after (codon-optimized) codon optimization using the GRCA tool. The graph shows the percentage distribution of codons that belong to a specific quality group. Codons with the most frequently used codon for a given amino acid in E. coli is assigned a value of 100. The codons which are distributed in codon quality groups having value ≤ 30 are used less frequently by that organism (threshold is shown as a dotted line) and are more likely to impede the expression. The presence of grey bars (i.e. non-optimized) with values ≤ 30 in the graph signified that it had codons that could affect its expression in E. coli. The absence of black bars (i.e. codon-optimized) with values ≤ 30 in the graph signified that it lacked codons that could affect its expression in E. coli.

12033_2021_305_MOESM2_ESM.tif

Supplementary file2 (TIF 2228 KB) Fig. S2 Evaluation of human SOX2 protein-coding sequence before (non-optimized) and after (codon-optimized) codon optimization using the GCUA 2.0 tool. The graph shows the codons of non-optimized and codon-optimized SOX2 coding sequence against the relative adaptiveness value. The relative adaptiveness value is based on the number of codons available concerning the organism for that particular amino acid. Therefore, for a given codon, relative adaptiveness value is determined by computing the multiplication of 100 with its usage frequency to the frequently used codon usage frequency. Codons with a relative adaptiveness value ≤ 30% (shown by arrows in black) are very likely to impede the expression in E. coli. The absence of grey bars in the codon-optimized graph shows that it was devoid of inhibitory codons.

Supplementary file3 (DOCX 14 KB)

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Thool, M., Dey, C., Bhattacharyya, S. et al. Generation of a Recombinant Stem Cell-Specific Human SOX2 Protein from Escherichia coli Under Native Conditions. Mol Biotechnol 63, 327–338 (2021). https://doi.org/10.1007/s12033-021-00305-y

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