Generic placeholder image

Protein & Peptide Letters


ISSN (Print): 0929-8665
ISSN (Online): 1875-5305

Research Article

Utilization of SUMO Tag and Freeze-thawing Method for a High-level Expression and Solubilization of Recombinant Human Angiotensinconverting Enzyme 2 (rhACE2) Protein in E. coli

Author(s): Mozafar Mohammadi*, Ramezan Ali Taheri, Peyman Bemani, Mohammad Sadegh Hashemzadeh, Gholamreza Farnoosh and Razieh Amini

Volume 29, Issue 7, 2022

Published on: 23 August, 2022

Page: [605 - 610] Pages: 6

DOI: 10.2174/0929866529666220715101357


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 the prophylaxis and treatment of COVID-19.

Objective: The recombinant human ACE2 (rhACE2) is prone to form an inclusion body when expressed in the bacterial cells.

Methods: We used the SUMO tag fused to the rhACE2 molecule to increase the expression level and solubility of the fusion protein. Afterward, the freeze-thawing method plus 2 M urea solubilized aggregated proteins. Subsequently, the affinity of solubilized rhACE2 to the receptor binding domain (RBD) of the SARS-CoV-2 spike was assayed by ELISA and SPR methods.

Results: SUMO protein succeeded in increasing the expression level but not solubilization of the fusion protein. The freeze-thawing method could solubilize and recover 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.

Keywords: ACE2, inclusion body, mild solubilization, RBD, SARS-CoV-2, SUMO tag, rhACE2.

Graphical Abstract
Zhou, P.; Yang, X-L.; Wang, X-G.; Hu, B.; Zhang, L.; Zhang, W.; Si, H.R.; Zhu, Y.; Li, B.; Huang, C.L.; Chen, H.D.; Chen, J.; Luo, Y.; Guo, H.; Jiang, R.D.; Liu, M.Q.; Chen, Y.; Shen, X.R.; Wang, X.; Zheng, X.S.; Zhao, K.; Chen, Q.J.; Deng, F.; Liu, L.L.; Yan, B.; Zhan, F.X.; Wang, Y.Y.; Xiao, G.F.; Shi, Z.L. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature, 2020, 579(7798), 270-273.
[] [PMID: 32015507]
He, J.; Tao, H.; Yan, Y. Molecular mechanism of evolution and human infection with the novel coronavirus (2019-nCoV). bioRxiv, 2020, 2020., 952903.
Glasgow, A.; Glasgow, J.; Limonta, D.; Solomon, P.; Lui, I.; Zhang, Y.; Nix, M.A.; Rettko, N.J.; Zha, S.; Yamin, R.; Kao, K.; Rosenberg, O.S.; Ravetch, J.V.; Wiita, A.P.; Leung, K.K.; Lim, S.A.; Zhou, X.X.; Hobman, T.C.; Kortemme, T.; Wells, J.A. Engineered ACE2 receptor traps potently neutralize SARS-CoV-2. Proc. Natl. Acad. Sci. USA, 2020, 117(45), 28046-28055.
[] [PMID: 33093202]
Malakhov, M.P.; Mattern, M.R.; Malakhova, O.A.; Drinker, M.; Weeks, S.D.; Butt, T.R. SUMO fusions and SUMO-specific protease for efficient expression and purification of proteins. J. Struct. Funct. Genomics, 2004, 5(1-2), 75-86.
[] [PMID: 15263846]
Marblestone, J.G.; Edavettal, S.C.; Lim, Y.; Lim, P.; Zuo, X.; Butt, T.R. Comparison of SUMO fusion technology with traditional gene fusion systems: Enhanced expression and solubility with SUMO. Protein Sci., 2006, 15(1), 182-189.
[] [PMID: 16322573]
Zuo, X.; Mattern, M.R.; Tan, R.; Li, S.; Hall, J.; Sterner, D.E.; Shoo, J.; Tran, H.; Lim, P.; Sarafianos, S.G.; Kazi, L.; Navas-Martin, S.; Weiss, S.R.; Butt, T.R. Expression and purification of SARS coronavirus proteins using SUMO-fusions. Protein Expr. Purif., 2005, 42(1), 100-110.
[] [PMID: 15939295]
Qi, X.; Sun, Y.; Xiong, S. A single freeze-thawing cycle for highly efficient solubilization of inclusion body proteins and its refolding into bioactive form. Microb. Cell Fact., 2015, 14, 24-24.
[] [PMID: 25879903]
Xiao, T.; Lu, J.; Zhang, J.; Johnson, R.I.; McKay, L.G.A.; Storm, N.; Lavine, C.L.; Peng, H.; Cai, Y.; Rits-Volloch, S.; Lu, S.; Quinlan, B.D.; Farzan, M.; Seaman, M.S.; Griffiths, A.; Chen, B. A trimeric human angiotensin-converting enzyme 2 as an anti-SARS-CoV-2 agent. Nat. Struct. Mol. Biol., 2021, 28(2), 202-209.
[] [PMID: 33432247]
Li, F.; Li, W.; Farzan, M.; Harrison, S.C. Structure of SARS coronavirus spike receptor-binding domain complexed with receptor. Science, 2005, 309(5742), 1864-1868.
[] [PMID: 16166518]
Kruse, R.L. Therapeutic strategies in an outbreak scenario to treat the novel coronavirus originating in Wuhan, China. F1000 Res., 2020, 9(72), 72.
[] [PMID: 32117569]
Haschke, M.; Schuster, M.; Poglitsch, M.; Loibner, H.; Salzberg, M.; Bruggisser, M.; Penninger, J.; Krähenbühl, S. Pharmacokinetics and pharmacodynamics of recombinant human angiotensin-converting enzyme 2 in healthy human subjects. Clin. Pharmacokinet., 2013, 52(9), 783-792.
[] [PMID: 23681967]
Khan, A.; Benthin, C.; Zeno, B.; Albertson, T.E.; Boyd, J.; Christie, J.D.; Hall, R.; Poirier, G.; Ronco, J.J.; Tidswell, M.; Hardes, K.; Powley, W.M.; Wright, T.J.; Siederer, S.K.; Fairman, D.A.; Lipson, D.A.; Bayliffe, A.I.; Lazaar, A.L. A pilot clinical trial of recombinant human angiotensin-converting enzyme 2 in acute respiratory distress syndrome. Crit. Care, 2017, 21(1), 234.
[] [PMID: 28877748]
Gao, X.; Liang, K.; Mei, S.; Peng, S.; Vong, E.G.; Zhan, J. An efficient system to generate truncated human angiotensin converting enzyme 2 (hACE2) capable of binding RBD and spike protein of SARS-CoV2. Protein Expr. Purif., 2021, 184, 105889.
[] [PMID: 33852951]
[15] Recombinant Human Angiotensin-converting Enzyme 2 (rhACE2) as a treatment for patients with COVID-19 (APN01-COVID-19). Available from: https://clinicaltrials. gov/ct2/show/NCT04335136
[16] Safety and tolerability study of APN01 (Recombinant Human Angiotensin Converting Enzyme 2). Available from:
Apeiron Biologics. APN01 – Our work on a potential drug candidate for COVID-19 treatment. Available from: 2019,
Zoufaly, A.; Poglitsch, M.; Aberle, J.H.; Hoepler, W.; Seitz, T.; Traugott, M.; Grieb, A.; Pawelka, E.; Laferl, H.; Wenisch, C.; Neuhold, S.; Haider, D.; Stiasny, K.; Bergthaler, A.; Puchhammer-Stoeckl, E.; Mirazimi, A.; Montserrat, N.; Zhang, H.; Slutsky, A.S.; Penninger, J.M. Human recombinant soluble ACE2 in severe COVID-19. Lancet Respir. Med., 2020, 8(11), 1154-1158.
[] [PMID: 33131609]
Monteil, V.; Kwon, H.; Prado, P.; Hagelkrüys, A.; Wimmer, R.A.; Stahl, M.; Leopoldi, A.; Garreta, E.; Hurtado Del Pozo, C.; Prosper, F.; Romero, J.P.; Wirnsberger, G.; Zhang, H.; Slutsky, A.S.; Conder, R.; Montserrat, N.; Mirazimi, A.; Penninger, J.M. Inhibition of SARS-CoV-2 infections in engineered human tissues using clinical-grade soluble human ACE2. Cell, 2020, 181(4), 905-913.e7.
[] [PMID: 32333836]
Müller, D.; Bayer, K.; Mattanovich, D. Potentials and limitations of prokaryotic and eukaryotic expression systems for recombinant protein production – a comparative view. Microb. Cell Fact., 2006, 5(1), 61.
Canaves, J.M.; Page, R.; Wilson, I.A.; Stevens, R.C. Protein biophysical properties that correlate with crystallization success in Thermotoga maritima: maximum clustering strategy for structural genomics. J. Mol. Biol., 2004, 344(4), 977-991.
[] [PMID: 15544807]
Goh, C.S.; Lan, N.; Douglas, S.M.; Wu, B.; Echols, N.; Smith, A.; Milburn, D.; Montelione, G.T.; Zhao, H.; Gerstein, M. Mining the structural genomics pipeline: Identification of protein properties that affect high-throughput experimental analysis. J. Mol. Biol., 2004, 336(1), 115-130.
[] [PMID: 14741208]
Pikal-Cleland, K.A.; Rodríguez-Hornedo, N.; Amidon, G.L.; Carpenter, J.F. Protein denaturation during freezing and thawing in phosphate buffer systems: Monomeric and tetrameric beta-galactosidase. Arch. Biochem. Biophys., 2000, 384(2), 398-406.
[] [PMID: 11368330]
Cao, E.; Chen, Y.; Cui, Z.; Foster, P.R. Effect of freezing and thawing rates on denaturation of proteins in aqueous solutions. Biotechnol. Bioeng., 2003, 82(6), 684-690.
[] [PMID: 12673768]
Ali, A.; Vijayan, R. Dynamics of the ACE2-SARS-CoV-2/SARSCoV spike protein interface reveal unique mechanisms. Sci. Rep., 2020, 10(1), 14214.
[] [PMID: 32848162]
Barton, M.I.; MacGowan, S.A.; Kutuzov, M.A.; Dushek, O.; Barton, G.J.; van der Merwe, P.A. Effects of common mutations in the SARS-CoV-2 Spike RBD and its ligand, the human ACE2 receptor on binding affinity and kinetics. eLife, 2021, 10, e70658.
[] [PMID: 34435953]

© 2023 Bentham Science Publishers | Privacy Policy