Utilization of SUMO tag and freeze-thawing method for high level expression and solubilization of recombinant human angiotensin-converting enzyme 2 (rhACE2) protein in E. coli

Abstract

BACKGROUND: SARS-CoV-2 uses angiotensin-converting enzyme 2 (ACE2) as a receptor for entering the host cells. Production of the ACE2 molecule is important because of its potency to use as a blocker and therapeutic agent against SARS-CoV-2 for prophylaxis and treatment of COVID-19. OBJECTIVE: The recombinant human ACE2 (rhACE2) prone to form inclusion body when it expressed in the bacterial cells. METHOD: We used SUMO tag fused to rhACE2 molecule to increase the expression level and solubility of the fusion protein. Afterward, aggregated proteins were solubilized using freeze-thawing method plus 2 M urea. Subsequently, affinity of solubilized rhACE2 to the receptor binding domain (RBD) of SARS-CoV-2 spike was assayed by ELISA and SPR methods. RESULTS: SUMO protein succeed to increase the level of expression but not solubilization of the fusion protein. The freeze-thawing method could solubilize and recovered the aggregated fusion proteins, significantly. Also, ELISA and SPR assays confirmed the interaction between solubilized rhACE2 and RBD with high affinity. CONCLUSION: The SUMO tag and freeze-thawing method would be utilized for high-level expression and solubilization of recombinant rhACE2 protein.