Optimization strategies for improving soluble expression of sars-cov-2 s1 protein using sumo-fusion technology

ABSTRACT

BACKGROUND AND AIM: E. coli are widely used for recombinant protein development, due to its low cost, ease of manipulation, and availability of well established molecular tools and techniques. Due to a lack of sophisticated machinery to undertake posttranslational modifications, the E. coli bacterial culture is limited in its ability to express more complex proteins, resulting in low solubility of the protein of interest that is generated as inclusion bodies. Although we were able to produce the recombinant SARS-CoV-2-S1 protein at high expression levels in our earlier investigation, we were also able to obtain nearly the whole protein as inclusion body. To overcome this problem, different solubility strategies have been tried. In this study, we developed an E.coli expression strategy based on the expression of the S1 protein as a fusion of SUMO fusion protein. METHODS: The DNA sequence of S1 protein was cloned into the pET SUMO expression vector, resulting in a construct expressing a N-terminal tag SUMO fusion protein. To achieve the high-level expression of S1, small scale expression conditions were optimized in E. coli BL21 (DE3) containing pET SUMO-S1 with different induction temperatures, times and IPTG concentrations. Additionally, different medium was also tested for the expression of S1 protein. For each parameter, solubility and expression of cell lysates from uninduced and induced cultures, plus the soluble and insoluble fractions from induced cultures were analyzed by SDS-PAGE and Western Blot. RESULTS: SDS-PAGE and Western Blot analysis showed the presence of a ∼83 kDa recombinant fusion protein. The maximum level of expression of the recombinant protein was observed at 30 , 4 h after induction with 0,55 mM IPTG. CONCLUSIONS: This study showed that the use of SUMO fusion tag partially increases the production of S1 protein in the form of soluble fractions and optimization studies continue.