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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.

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.