重组人ACE2催化亚基与霍乱毒素B亚基杂交蛋白在原核宿主系统中的高效表达、纯化及结构分析

IF 1.9 4区 生物学 Q4 BIOCHEMISTRY & MOLECULAR BIOLOGY The Protein Journal Pub Date : 2023-11-28 DOI:10.1007/s10930-023-10164-y
Maryam Ghahramani, Mohammad Bagher Shahsavani, Seyed Hossein Khaleghinejad, Ali Niazi, Ali Akbar Moosavi-Movahedi, Reza Yousefi
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摘要

血管紧张素转换酶2 (ACE2)与冠状病毒刺突蛋白具有特异性相互作用,使其能够进入人体细胞。这种膜酶将血管紧张素II转化为血管紧张素1-7,对保护心脏和改善肺功能具有重要作用。人类重组ACE2 (hrACE2)具有许多治疗特性,特别是在对抗糖尿病和高血压相关并发症以及防止冠状病毒进入靶组织方面。在本研究中,我们为含有ACE2催化亚基和霍乱毒素B亚基的杂交蛋白(CTB-ACE2)设计了一个合适的基因结构。这种结构特征可能有助于重组杂交蛋白进入粘膜组织,包括肺组织。研究了该杂交蛋白在BL21细菌宿主细胞中的表达优化。用ELISA法对该杂交蛋白进行鉴定。在0.25 mM IPTG和1%蔗糖的作用下诱导10 h后,大量杂交蛋白(分子量约100 kDa)被表达为包涵体。最后,采用多种生物物理方法评估蛋白质的结构特征。CTB-ACE2的荧光发射强度和寡聚体大小分布显示温度依赖性变化。在杂交蛋白的结构中,β-片和α-螺旋也占主导地位,该蛋白也表现出良好的化学稳定性。总之,根据我们的研究结果,CTB-ACE2蛋白的高效表达和成功纯化可能为其在covid-19、糖尿病和高血压等疾病的治疗应用铺平道路。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

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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)

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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来源期刊
The Protein Journal
The Protein Journal 生物-生化与分子生物学
CiteScore
5.20
自引率
0.00%
发文量
57
审稿时长
12 months
期刊介绍: The Protein Journal (formerly the Journal of Protein Chemistry) publishes original research work on all aspects of proteins and peptides. These include studies concerned with covalent or three-dimensional structure determination (X-ray, NMR, cryoEM, EPR/ESR, optical methods, etc.), computational aspects of protein structure and function, protein folding and misfolding, assembly, genetics, evolution, proteomics, molecular biology, protein engineering, protein nanotechnology, protein purification and analysis and peptide synthesis, as well as the elucidation and interpretation of the molecular bases of biological activities of proteins and peptides. We accept original research papers, reviews, mini-reviews, hypotheses, opinion papers, and letters to the editor.
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