Artificial Lipid Biomembranes for Full‐Length SARS‐CoV‐2 Receptor

Abstract

The angiotensin-converting enzyme 2 (ACE2), as a functional receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is essential for assessing potential hosts and treatments. However, many studies are based on its truncated version but not full-length structure. Indeed, a single transmembrane (TM) helix presents in the full-length ACE2, influencing its interaction with SARS-CoV-2. Therefore, synthesis of the full-length ACE2 is an urgent requirement. Here, cell-free membrane protein synthesis systems (CFMPSs) are constructed for full-length membrane proteins. MscL is screened as a model among ten membrane proteins based on their expression and solubility. Next, CFMPSs are constructed and optimized based on natural vesicles, vesicles with four membrane proteins removed or two chaperonins added, and 37 types of nanodiscs. They all increase membrane protein solubility to over 50%. Finally, the full-length ACE2 of 21 species are successfully expressed with yields between 0.4 and 0.9 mg mL −1 . The definite functional differences from the truncated version suggest that the TM region affects ACE2's structure and function. CFMPSs can be extended to more membrane proteins, paving the way for further applications.

Toward Minimal Transcription–Translation Machinery

“…Therefore, to test the effect of GRACE systems based on different chassis on the expression of membrane protein, two genome-reduced strains were applied to the cellfree system for membrane protein expression. CCR5, a typical membrane protein with 7-segment transmembrane region, 28 was selected as a model membrane protein for the test. Membrane protein CCR5 showed dual advantages in terms of expression and solubility in the GRACE system.…”

“…CFTT was first used by Borsook and Winnick to study animal tissue homogenates and how amino acids are incorporated into proteins in the 1950s . Till now, CFTT has also been used in various basic protein studies, including crystallization and structural studies. , In terms of engineering applications, CFTT has a convenient form of high-throughput screening, accelerated design-build-test-learn cycle, high synthesis rate and product yield, which make it a significant advantage on an industrial scale . Therefore, CFTT has been used to produce high-value small molecule products, biologics, and special proteins. , Continued research in the area of scaling up will have the potential to accelerate industrial adaptation.…”