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).

Autor: Ghahramani M; Protein Chemistry Laboratory (PCL), Department of Biology, College of Sciences, Shiraz University, Shiraz, Iran., Shahsavani MB; Protein Chemistry Laboratory (PCL), Department of Biology, College of Sciences, Shiraz University, Shiraz, Iran., Khaleghinejad SH; Protein Chemistry Laboratory (PCL), Department of Biology, College of Sciences, Shiraz University, Shiraz, Iran., Niazi A; Institute of Biotechnology, Shiraz University, Shiraz, Iran., Moosavi-Movahedi AA; Institute of Biochemistry and Biophysics (IBB), University of Tehran, Tehran, Iran., Yousefi R; Protein Chemistry Laboratory (PCL), Department of Biology, College of Sciences, Shiraz University, Shiraz, Iran. ryousefi@shirazu.ac.ir.; Institute of Biochemistry and Biophysics (IBB), University of Tehran, Tehran, Iran. ryousefi@shirazu.ac.ir.
Jazyk: angličtina
Zdroj: The protein journal [Protein J] 2024 Feb; Vol. 43 (1), pp. 24-38. Date of Electronic Publication: 2023 Nov 28.
DOI: 10.1007/s10930-023-10164-y
Abstrakt: 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.
(© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)
Databáze: MEDLINE
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