Laboratory for Stem Cell Engineering and Regenerative Medicine, Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, Assam, 781039, India.
Department of Biotechnology, National Institute of Pharmaceutical Education and Research Guwahati, Changsari, Guwahati, Assam, 781101, India.
Mol Biotechnol. 2021 Apr;63(4):327-338. doi: 10.1007/s12033-021-00305-y. Epub 2021 Feb 11.
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.
干细胞特异性 SOX2 转录因子对于早期胚胎发育以及胚胎和神经干细胞特性的维持至关重要。它对于诱导多能干细胞和神经干细胞的生成也至关重要，从而为患者特异性治疗提供了广阔的前景。在这里，我们报告了在细菌系统中从天然条件下可溶性表达和纯化人 SOX2 蛋白。为了生成这种大分子，我们对蛋白质编码序列进行了密码子优化，并将其与核定位信号、蛋白转导结构域和 His 标签融合。然后将其克隆到蛋白表达载体中，并在大肠杆菌中表达。随后，我们筛选并确定了最佳表达条件，以获得可溶性形式的重组融合蛋白，并研究了融合标签在两端位置对其表达的影响。此外，我们在天然条件下将两种版本的重组 SOX2 融合蛋白纯化至均一性，并证明它们保持了其二级结构。这种分子工具可以替代 SOX2 的遗传和病毒形式，以促进无整合的诱导多能干细胞和神经干细胞的衍生。此外，它还可以用于阐明其在干细胞、各种细胞过程和疾病中的作用，以及用于结构和生化研究。
