In a groundbreaking development, scientists from the U.S. Department of Energy's Brookhaven National Laboratory and Columbia University have successfully produced significant quantities of the receptor protein that SARS-CoV-2, the virus responsible for COVID-19, uses to bind to human cells.
The team's primary objective at the onset of the pandemic was to generate large amounts of the human ACE2 protein and attach it to nanoparticles. These ACE2-coated nanoparticles were intended for testing as potential anti-viral therapeutics or as sensors for virus detection. "For either of these applications, you need large quantities of protein, and the protein has to be fully functional," explained Paul Freimuth, a virologist at Brookhaven Lab, who spearheaded the research.
Producing functional membrane proteins like ACE2 is notably challenging due to the complex process of protein localization in the cell membrane. These proteins undergo various modifications, including the addition of carbohydrate molecules, which are crucial for their folding into the final 3D structure and functionality in the membrane. "Carbohydrates account for about one-third of the mass of ACE2 protein," Freimuth noted.
To overcome the limitations of simpler cells like bacteria, which lack the necessary enzymes for these modifications, the team turned to mouse cells. These cells, being mammalian, can process carbohydrates similarly to human cells. Moreover, mouse cells are adept at incorporating and expressing foreign genes. Importantly, the mouse version of the ACE2 protein does not bind to the SARS-CoV-2 spike, providing a clear indicator of successful human ACE2 protein production in the mouse cells.
The researchers utilized the intact human ACE2 gene, including its regulatory information, to increase the likelihood of correct gene incorporation and expression in mouse cells. They then exposed mouse cells to nanoparticles coated with this DNA fragment and a gene for antibiotic resistance. The cells that successfully integrated the foreign genes and expressed the antibiotic resistance survived and were expanded into individual cultures for further testing.
Approximately 70% of these antibiotic-resistant colonies expressed the human ACE2 protein on their surface. Further analysis revealed an average of 28 copies of the human ACE2 gene in these colonies. The mouse cells retained these foreign gene copies and continued producing the human ACE2 protein for at least 90 cell generations. Some mouse cell clones produced about 50 times more ACE2 than typically present on mouse cells.
The functionality of the mouse-made human ACE2 proteins was confirmed through various tests, including their ability to bind to a pseudovirus containing the COVID spike protein. "These infectivity assays showed that the human ACE2 protein expressed on these mouse cells is fully functional," Freimuth affirmed.
This research not only paves the way for potential applications of recombinant ACE2 protein but also demonstrates a novel method for producing a wide range of complex proteins.
These include cell-surface receptors involved in numerous biological and disease processes, as well as industrially significant proteins like monoclonal antibodies and enzymes. "Our method of using intact genes along with mouse cells that can be adapted to grow in huge suspension cultures could advance the large-scale production of these and other important proteins," Freimuth concluded.