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Efficient Expression in the Prokaryotic Host System, Purification and Structural Analyses of the Recombinant Human ACE2 Catalytic Subunit as a Hybrid Protein with the B Subunit of Cholera Toxin (CTB-ACE2)

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Abstract

Angiotensin-converting enzyme 2 (ACE2) has a specific interaction with the coronavirus spike protein, enabling its entry into human cells. This membrane enzyme converts angiotensin II into angiotensin 1–7, which has an essential role in protecting the heart and improving lung function. Many therapeutic properties have been attributed to the human recombinant ACE2 (hrACE2), especially in combating complications related to diabetes mellitus and hypertension, as well as, preventing the coronavirus from entering the target tissues. In the current study, we designed an appropriate gene construct for the hybrid protein containing the ACE2 catalytic subunit and the B subunit of cholera toxin (CTB-ACE2). This structural feature will probably help the recombinant hybrid protein enter the mucosal tissues, including the lung tissue. Optimization of this hybrid protein expression was investigated in BL21 bacterial host cells. Also, the hybrid protein was identified with an appropriate antibody using the ELISA method. A large amount of the hybrid protein (molecular weight of ~ 100 kDa) was expressed as the inclusion body when the induction was performed in the presence of 0.25 mM IPTG and 1% sucrose for 10 h. Finally, the protein structural features were assessed using several biophysical methods. The fluorescence emission intensity and oligomeric size distribution of the CTB-ACE2 suggested a temperature-dependent alteration. The β-sheet and α-helix were also dominant in the hybrid protein structure, and this protein also displays acceptable chemical stability. In overall, according to our results, the efficient expression and successful purification of the CTB-ACE2 protein may pave the path for its therapeutic applications against diseases such as covid-19, diabetes mellitus and hypertension.

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References

  1. Atlas SA (2007) The renin-angiotensin aldosterone system: pathophysiological role and pharmacologic inhibition. J Manag Care Pharm JMCP 13:9–20. https://doi.org/10.18553/jmcp.2007.13.s8-b.9

    Article  PubMed  Google Scholar 

  2. Verma A, Xu K, Du T, Zhu P, Liang Z, Liao S, Zhang J, Raizada MK, Grant MB, Li Q (2019) Expression of human ACE2 in Lactobacillus and Beneficial effects in diabetic retinopathy in mice. Mol Ther Methods Clin Dev 14:161–170. https://doi.org/10.1016/j.omtm.2019.06.007

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Hamming I, Timens W, Bulthuis MLC, Lely AT, Navis GJ, van Goor H (2004) Tissue distribution of ACE2 protein, the functional receptor for SARS coronavirus. A first step in understanding SARS pathogenesis. J Pathol 203:631–637. https://doi.org/10.1002/path.1570

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Lippi G, Lavie CJ, Henry BM, Sanchis-Gomar F (2020) Do genetic polymorphisms in angiotensin converting enzyme 2 (ACE2) gene play a role in coronavirus disease 2019 (COVID-19)? Clin Chem Lab Med 58(2020):1415–1422. https://doi.org/10.1515/cclm-2020-0727

    Article  CAS  PubMed  Google Scholar 

  5. Lambert DW, Yarski M, Warner FJ, Thornhill P, Parkin ET, Smith AI, Hooper NM, Turner AJ (2005) Tumor necrosis factor-alpha convertase (ADAM17) mediates regulated ectodomain shedding of the severe-acute respiratory syndrome-coronavirus (SARS-CoV) receptor, angiotensin-converting enzyme-2 (ACE2). J Biol Chem 280:30113–30119. https://doi.org/10.1074/jbc.M505111200

    Article  CAS  PubMed  Google Scholar 

  6. Aragão DS, Cunha TS, Arita DY, Andrade MCC, Fernandes AB, Watanabe IKM, Mortara RA, Casarini DE (2011) Purification and characterization of angiotensin converting enzyme 2 (ACE2) from murine model of mesangial cell in culture. Int J Biol Macromol 49:79–84. https://doi.org/10.1016/j.ijbiomac.2011.03.018

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Tipnis SR, Hooper NM, Hyde R, Karran E, Christie G, Turner AJ (2000) A human homolog of angiotensin-converting enzyme. Cloning and functional expression as a captopril-insensitive carboxypeptidase. J Biol Chem 275:33238–33243. https://doi.org/10.1074/jbc.M002615200

    Article  CAS  PubMed  Google Scholar 

  8. Crackower MA, Sarao R, Oudit GY, Yagil C, Kozieradzki I, Scanga SE, Oliveira-dos-Santos AJ, da Costa J, Zhang L, Pei Y, Scholey J, Ferrario CM, Manoukian AS, Chappell MC, Backx PH, Yagil Y, Penninger JM (2002) Angiotensin-converting enzyme 2 is an essential regulator of heart function. Nature 417:822–828. https://doi.org/10.1038/nature00786

    Article  ADS  CAS  PubMed  Google Scholar 

  9. Xiao H, Nie X-T, Ji X-X, Yan S, Zhu B, Zhang Y-S (2020) Establishing prokaryotic expression system of angiotensin-converting enzyme 2 (ACE2) gene in pigs. BioRxiv. https://doi.org/10.1101/2020.03.12.988634

    Article  PubMed  PubMed Central  Google Scholar 

  10. Ghahramani M, Yousefi R, Niazi A, Kurganov B (2020) The congenital cataract-causing mutations P20R and A171T are associated with important changes in the amyloidogenic feature, structure and chaperone-like activity of human αB-crystallin. Biopolymers 111:e23350

    Article  CAS  PubMed  Google Scholar 

  11. Teale FWJ, Weber G (1957) Ultraviolet fluorescence of the aromatic amino acids. Biochem J 65:476

    Article  CAS  PubMed  Google Scholar 

  12. Zhang Y-Z, Zhou B, Liu Y-X, Zhou C-X, Ding X-L, Liu Y (2008) Fluorescence study on the interaction of bovine serum albumin with p-aminoazobenzene. J Fluoresc 18:109–118

    Article  CAS  PubMed  Google Scholar 

  13. Khoshaman K, Oryan A, Moosavi-Movahedi AA, Tamaddon AM, Kurganov BI, Yousefi R, Abolmaali SS (2017) impact of different mutations at Arg54 on structure, chaperone-like activity and oligomerization state of human αA-crystallin: The pathomechanism underlying congenital cataract-causing mutations R54L, R54P and R54C. BBA-Proteins Proteom 1865:604–618

    Article  CAS  Google Scholar 

  14. Maltas E (2014) Binding interactions of niclosamide with serum proteins. J Food Drug Anal 22:549–555

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Sharma KK, Kumar GS, Murphy AS, Kester K (1998) Identification of 1, 1′-bi (4-anilino) naphthalene-5, 5′-disulfonic acid binding sequences in α-crystallin. J Biol Chem 273:15474–15478

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Bortolotti A, Wong YH, Korsholm SS, Bahring NHB, Bobone S, Tayyab S, van de Weert M, Stella L (2016) On the purported “backbone fluorescence” in protein three-dimensional fluorescence spectra. Rsc Adv 6:112870–112876

    Article  ADS  CAS  Google Scholar 

  17. Grigoryan KR, Shilajyan HA (2017) Fluorescence 2D and 3D spectra analysis of tryptophan, tyrosine and phenylalanine. Chem Biol 51:3–7

    Google Scholar 

  18. Whitmore L, Wallace BA (2004) DICHROWEB, an online server for protein secondary structure analyses from circular dichroism spectroscopic data. Nucleic Acids Res 32:W668–W673

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Sadat A, Joye IJ (2020) Peak fitting applied to Fourier transform infrared and Raman spectroscopic analysis of proteins. Appl Sci 10:5918

    Article  CAS  Google Scholar 

  20. Khaleghinejad SH, Shahsavani MB, Ghahramani M, Yousefi R (2023) Investigating the role of double mutations R12C/P20R, and R12C/R69C on structure, chaperone-like activity, and amyloidogenic properties of human αB-crystallin. Int J Biol Macromol 242:124590

    Article  CAS  PubMed  Google Scholar 

  21. Zhou C, Qi W, Lewis EN, Carpenter JF (2015) Concomitant Raman spectroscopy and dynamic light scattering for characterization of therapeutic proteins at high concentrations. Anal Biochem 472:7–20

    Article  CAS  PubMed  Google Scholar 

  22. Schägger H, Von Jagow G (1987) Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166:368–379

    Article  PubMed  Google Scholar 

  23. Gasteiger E, Hoogland C, Gattiker A, et al (2005) Protein identification and analysis tools on the ExPASy server. Springer

    Article  PubMed  Google Scholar 

  24. Hawe A, Sutter M, Jiskoot W (2008) Extrinsic fluorescent dyes as tools for protein characterization. Pharm Res 25:1487–1499

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Montgomerie S, Cruz JA, Shrivastava S, Arndt D, Berjanskii M, Wishart DS (2008) PROTEUS2: a web server for comprehensive protein structure prediction and structure-based annotation. Nucleic Acids Res 36:W202–W209. https://doi.org/10.1093/nar/gkn255

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Bursell S-E, Yu N-T (1990) Fluorescence and Raman spectroscopy of the crystalline lens. Noninvasive Diagn Tech Ophthalmol 319–341

  27. Rygula A, Majzner K, Marzec KM, Kaczor A, Pilarczyk M, Baranska M (2013) Raman spectroscopy of proteins: a review. J Raman Spectrosc 44:1061–1076

    Article  ADS  CAS  Google Scholar 

  28. Benevides JM, Overman SA, Thomas GJ Jr (2003) Raman spectroscopy of proteins. Curr Protoc Protein Sci 33:17–18

    Article  Google Scholar 

  29. Wen Z (2007) Raman spectroscopy of protein pharmaceuticals. J Pharm Sci 96:2861–2878

    Article  CAS  PubMed  Google Scholar 

  30. Lubbe L, Cozier GE, Oosthuizen D, Acharya KR, Sturrock ED (2020) ACE2 and ACE: structure-based insights into mechanism, regulation and receptor recognition by SARS-CoV. Clin Sci Lond Engl 1979 134:2851–2871. https://doi.org/10.1042/CS20200899

    Article  CAS  Google Scholar 

  31. Zhang H, Baker A (2017) Recombinant human ACE2: acing out angiotensin II in ARDS therapy. Crit Care Lond Engl 21:305. https://doi.org/10.1186/s13054-017-1882-z

    Article  Google Scholar 

  32. Oudit GY, Liu GC, Zhong J, Basu R, Chow FL, Zhou J, Loibner H, Janzek E, Schuster M, Penninger JM, Herzenberg AM, Kassiri Z, Scholey JW (2010) Human recombinant ACE2 reduces the progression of diabetic nephropathy. Diabetes 59:529–538. https://doi.org/10.2337/db09-1218

    Article  CAS  PubMed  Google Scholar 

  33. Tikellis C, Thomas MC (2012) Angiotensin-converting enzyme 2 (ACE2) is a key modulator of the renin angiotensin system in health and disease. Int J Pept 2012:256294. https://doi.org/10.1155/2012/256294

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Imai Y, Kuba K, Rao S, Huan Y, Guo F, Guan B, Yang P, Sarao R, Wada T, Leong-Poi H, Crackower MA, Fukamizu A, Hui C-C, Hein L, Uhlig S, Slutsky AS, Jiang C, Penninger JM (2005) Angiotensin-converting enzyme 2 protects from severe acute lung failure. Nature 436:112–116. https://doi.org/10.1038/nature03712

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  35. Zhang H, Penninger JM, Li Y, Zhong N, Slutsky AS (2020) Angiotensin-converting enzyme 2 (ACE2) as a SARS-CoV-2 receptor: molecular mechanisms and potential therapeutic target. Intensive Care Med 46:586–590. https://doi.org/10.1007/s00134-020-05985-9

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Tada T, Fan C, Chen JS, Kaur R, Stapleford KA, Gristick H, Dcosta BM, Wilen CB, Nimigean CM, Landau NR (2020) An ACE2 microbody containing a single immunoglobulin fc domain is a potent inhibitor of SARS-CoV-2. Cell Rep 33:108528. https://doi.org/10.1016/j.celrep.2020.108528

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. D’Onofrio N, Scisciola L, Sardu C, Trotta MC, De Feo M, Maiello C, Mascolo P, De Micco F, Turriziani F, Municinò E, Monetti P, Lombardi A, Napolitano MG, Marino FZ, Ronchi A, Grimaldi V, Hermenean A, Rizzo MR, Barbieri M, Franco R, Pietro Campobasso C, Napoli C, Municinò M, Paolisso G, Balestrieri ML, Marfella R (2021) Glycated ACE2 receptor in diabetes: open door for SARS-COV-2 entry in cardiomyocyte. Cardiovasc Diabetol 20:99. https://doi.org/10.1186/s12933-021-01286-7

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Towler P, Staker B, Prasad SG, Menon S, Tang J, Parsons T, Ryan D, Fisher M, Williams D, Dales NA, Patane MA, Pantoliano MW (2004) ACE2 X-ray structures reveal a large hinge-bending motion important for inhibitor binding and catalysis. J Biol Chem 279:17996–18007. https://doi.org/10.1074/jbc.M311191200

    Article  CAS  PubMed  Google Scholar 

  39. Chaudhuri K, Chatterjee SN (2009) Cholera toxin (CT): structure. Cholera toxins. Springer, Berlin, pp 105–123

    Chapter  Google Scholar 

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Acknowledgements

The financial support of the National Institute of Genetic Engineering and Biotechnology (NIGEB) is greatly appreciated. Also, the authors appreciate and acknowledge the financial support of the Shiraz University Research Council, and Iran National Science Foundation (INSF).

Funding

This work was also partially supported by INSF (grant number 99032483).

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Authors and Affiliations

  1. Protein Chemistry Laboratory (PCL), Department of Biology, College of Sciences, Shiraz University, Shiraz, Iran

    Maryam Ghahramani, Mohammad Bagher Shahsavani, Seyed Hossein Khaleghinejad & Reza Yousefi

  2. Institute of Biotechnology, Shiraz University, Shiraz, Iran

    Ali Niazi

  3. Institute of Biochemistry and Biophysics (IBB), University of Tehran, Tehran, Iran

    Ali Akbar Moosavi-Movahedi & Reza Yousefi

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  1. Maryam Ghahramani
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Contributions

MG: Software, Formal analysis, Investigation, Data Curation, Writing-Original Draft. MBS: Writing and Editing, Software, Investigation, Data Curation. SHK: Software, Investigation, Data Curation. AN: Methodology, Investigation Validation, Data Curation, Resources. AAM-Movahedi: Writing-Review and Editing, Visualization, Supervision, Funding acquisition. RY: Conceptualization, Methodology, Validation, Resources, Writing—Review and Editing, Visualization, Supervision, Project administration, Funding acquisition.

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Correspondence to Reza Yousefi.

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Ghahramani, M., Shahsavani, M.B., Khaleghinejad, S.H. et al. Efficient Expression in the Prokaryotic Host System, Purification and Structural Analyses of the Recombinant Human ACE2 Catalytic Subunit as a Hybrid Protein with the B Subunit of Cholera Toxin (CTB-ACE2). Protein J 43, 24–38 (2024). https://doi.org/10.1007/s10930-023-10164-y

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