Recombinant Engineering of SARS-CoV-2 Spike and N proteins

我们在这个项目中的目标是探索已发表的SARS-CoV-2刺突蛋白的结构，重点是其受体结合结构域（RBD），努力产生热稳定性增加的重组分子。为此，我们已经研究了已发表的结构和生物信息学已经作出了一些预测的定点突变体，可能会产生分子的稳定性增加。初步的研究已经证实了我们从E.大肠杆菌表达载体，以通过多个色谱步骤产生高纯度的分子，并证实其结合特异性抗体的能力。突变研究正在进行中。此外，我们已经获得了用于表达全长Spike蛋白及其管腔结构域的载体，并且正在哺乳动物系统中进行表达研究。此外，我们正在重新设计血管紧张素转换酶-2（ACE-2）受体，用于类似的表达、结合和诱变研究。

Our goal in this project is to explore published structures of the SARS-CoV-2 Spike protein with a focus on its receptor binding domain (RBD), in efforts to generate recombinant molecules of increased thermal stability. To this end we have examined published structures and bioinformatically have made a number of predictions of site-directed mutants that may yield molecules of increased stability. Initial studies have confirmed our ability to produce the wild type RBD in good yield from E. coli expression vectors, to produce the molecule in high purity through multiple chromatographic steps, and to confirm its ability to bind specific antibodies. Mutational studies are in progress. In addition, we have obtained vectors for the expression of the full-length Spike protein and its lumens domain, and expression studies in mammalian systems are underway. Also, we are reengineering the angiotensin converting enzyme-2 (ACE-2) receptor for similar expression, binding, and mutagenesis studies.