Molecular dynamics based improvement of the solubilizing self-cleavable tag Zbasic-ΔI-CM application in the preparation of recombinant proteins in Escherichia coli.

Zbasic-ΔI-CM is a novel intein-based self-cleavable tag we developed to accelerate the soluble expression of recombinant proteins in Escherichia coli (E. coli). Previously we found that intein activity could be interfered by its flanking exteins, and thus reducing the production efficiency and final yield. In this work, we used CXC-chemokine 9 (CXCL9) as a model C-extein, which fusion with Zbasic-ΔI-CM showed high intein activity. When the fusion protein got soluble expression, CXCL9 was released immediately and purified directly from cell lysis supernatant. The results demonstrated that Zbasic-ΔI-CM tag had successfully mediated the efficient production of high-quality CXCL9 with reduced time and resources consumption in comparison with inclusion bodies expression. Molecular dynamics simulations suggested that the improved cleavage activity of Zbasic-ΔI-CM upon fusion with CXCL9 may be due to the higher dynamics of the first half loop and stabilization of the second half loop of intein. Our results proved that the self-cleavable Zbasic-ΔI-CM mediated soluble expression could be a feasible process for cytokines like CXCL9, thus of attractive potentials for production of therapeutic proteins using E. coli expression system.