Prokaryotic Expression and Purification of the hSox2-HMG Domain.

The Sox (SRY-related HMG-box) protein family plays a crucial role in cellular differentiation, development, and gene regulation, with the HMG (high-mobility group) domain responsible for DNA binding and transcriptional regulation. Proteins in the SOX gene family contain an HMG domain that shares 50% homology with the HMG domain of the sex-determining factor SRY gene. The SOX gene family comprises 30 proteins, which are classified into 10 groups (A-H). As a member of this family, hSox2 has been shown to be involved in various biological processes, but its specific function remains unclear. Previous studies have used eukaryotic expression systems, GST-tag purification, and bacterial inclusion body refolding techniques to produce Sox family proteins. However, these methods are often limited by issues such as low yield, incorrect folding, or inefficient purification, restricting their application in functional and structural studies. In this study, a prokaryotic expression system for the hSox2-HMG domain was constructed using the pET22b vector and BL21(DE3) as the host strain. Protein expression was induced by IPTG, and initial purification was performed using Ni-NTA affinity chromatography, followed by ultrafiltration concentration and size exclusion chromatography to improve purity. By optimizing lysis and elution conditions, we successfully obtained hSox2-HMG protein with high expression levels and purity. This method provides a cost-effective and scalable strategy for hSox2-HMG production, ensuring high purity and correct folding of the protein. The optimized experimental protocol lays a foundation for structural and functional studies of hSox2-HMG. Key features • The hSox2-HMG protein was expressed in BL21(DE3) using the pET22b vector and IPTG induction, resulting in high-yield recombinant protein. • Ni-NTA affinity chromatography was employed for protein purification and combined with ultrafiltration concentration and size exclusion chromatography to enhance purity and ensure correct folding. • The established workflow provides an efficient, cost-effective, and scalable strategy for hSox2-HMG production, suitable for structural and functional studies. • The purified hSox2-HMG protein maintains structural integrity and can be used for further investigation of DNA-binding properties and regulatory functions.