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

Overview
Journal Mol Biotechnol
Publisher Springer
Date 2021 Feb 11
PMID 33570706
Citations 6
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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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References
1.
Takahashi K, Yamanaka S . Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006; 126(4):663-76. DOI: 10.1016/j.cell.2006.07.024. View

2.
Yu J, Vodyanik M, Smuga-Otto K, Antosiewicz-Bourget J, Frane J, Tian S . Induced pluripotent stem cell lines derived from human somatic cells. Science. 2007; 318(5858):1917-20. DOI: 10.1126/science.1151526. View

3.
Rowe R, Daley G . Induced pluripotent stem cells in disease modelling and drug discovery. Nat Rev Genet. 2019; 20(7):377-388. PMC: 6584039. DOI: 10.1038/s41576-019-0100-z. View

4.
Haridhasapavalan K, Borgohain M, Dey C, Saha B, Narayan G, Kumar S . An insight into non-integrative gene delivery approaches to generate transgene-free induced pluripotent stem cells. Gene. 2018; 686:146-159. DOI: 10.1016/j.gene.2018.11.069. View

5.
Borgohain M, Haridhasapavalan K, Dey C, Adhikari P, Thummer R . An Insight into DNA-free Reprogramming Approaches to Generate Integration-free Induced Pluripotent Stem Cells for Prospective Biomedical Applications. Stem Cell Rev Rep. 2018; 15(2):286-313. DOI: 10.1007/s12015-018-9861-6. View