Mohammadi Mozafar, Taheri Ramezan Ali, Bemani Peyman, Hashemzadeh Mohammad Sadegh, Farnoosh Gholamreza, Amini Razieh
Applied Biotechnology Research Centre, Baqiyatallah University of Medical Sciences, Tehran, Iran.
Nanobiotechnology Research Centre, Baqiyatallah University of Medical Sciences, Tehran, Iran.
Protein Pept Lett. 2022;29(7):605-610. doi: 10.2174/0929866529666220715101357.
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.
The recombinant human ACE2 (rhACE2) is prone to form an inclusion body when expressed in the bacterial cells.
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.
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.
The SUMO tag and freeze- Conclusion: The SUMO tag and freeze-thawing method would be utilized for high-level expression and solubilization of recombinant rhACE2 protein.
严重急性呼吸综合征冠状病毒2（SARS-CoV-2）利用血管紧张素转换酶2（ACE2）作为进入宿主细胞的受体。ACE2分子的产生很重要，因为它有潜力用作针对SARS-CoV-2的阻滞剂和治疗剂，用于预防和治疗2019冠状病毒病（COVID-19）。
重组人ACE2（rhACE2）在细菌细胞中表达时容易形成包涵体。
我们使用与rhACE2分子融合的小泛素样修饰蛋白（SUMO）标签来提高融合蛋白的表达水平和溶解度。之后，采用冻融法加2 M尿素溶解聚集蛋白。随后，通过酶联免疫吸附测定（ELISA）和表面等离子体共振（SPR）方法检测溶解的rhACE2与SARS-CoV-2刺突蛋白受体结合域（RBD）的亲和力。
SUMO蛋白成功提高了融合蛋白的表达水平，但未提高其溶解度。冻融法可显著溶解并回收聚集的融合蛋白。此外，ELISA和SPR检测证实溶解的rhACE2与RBD之间具有高亲和力的相互作用。
SUMO标签和冻融法将用于重组rhACE2蛋白的高水平表达和溶解。
