The Influence of IF۲ and SUMO Fusion Tags on the Expression and Solubility of the SpCas۹ Protein

Introduction: The expression of the Cas۹ protein in the prokaryotic host Escherichia coli is often associated with the formation of inclusion bodies. This study aimed to express and purify the Cas۹ protein in large quantities and soluble form in E. coli BL۲۱ (DE۳) for use in the CRISPR/Cas۹ system. Due to the large size of Cas۹, two solubility-enhancing fusion partners with relatively low molecular weights, initiation factor ۲ (IF۲) and small ubiquitin-like modifier (SUMO), were utilized. Methods: The IF۲ and SUMO gene sequences, ۴۸۰ and ۳۰۰ bp in length, respectively, were amplified by PCR and cloned into the pET-۲۱a-۳xNLS-SpCas۹ vector. The expression of both recombinant proteins, with approximate molecular weights of ۱۷۶ and ۱۶۶ kDa, was confirmed by SDS-PAGE and Western blotting. Protein purification was performed using Ni-NTA affinity chromatography. Results: The presence of IF۲ as a fusion partner increased the percentage of Cas۹ detected in the supernatant from ۵.۵۱% to ۶.۹۹%. Moreover, under optimal cultivation conditions, the recombinant IF۲-Cas۹ protein was produced in greater quantities than Cas۹ alone (۱۸.۵۰ versus ۱۳.۶۵ mg/L). The solubility analysis revealed that SUMO-Cas۹ exhibited higher solubility (۹.۲۵%) than Cas۹ alone (۵.۵۱%), representing a ۶۷.۹ ± ۸.۲% increase in soluble expressio. Conclusion: While both fusion partners enhanced Cas۹ expression in E. coli, SUMO demonstrated superior solubility-enhancing capability compared to IF۲; however, the functional activity of the purified protein remains to be confirmed.

کلیدواژه ها:

SpCas۹ expression ، CRISPR/Cas۹ ، Initiation factor ۲ ، Solubility-enhancing tag ، Small ubiquitin-like modifier ، Escherichia coli