Soluble SARS‐CoV‐2 RBD and human ACE2 peptidase domain produced in DrosophilaS2 cells show functions evoking virus–cell interface

Abstract

The interaction between the receptor‐binding domain (RBD) of the spike glycoprotein of SARS‐CoV‐2 and the peptidase domain of the human angiotensin‐converting enzyme 2 (ACE2) allows the first specific contact at the virus–cell interface making it the main target of neutralizing antibodies. Here, we show a unique and cost‐effective protocol using Drosophila S2 cells to produce both RBD and soluble human ACE2 peptidase domain (shACE2) as thermostable proteins, purified via Strep‐tag with yields >40 mg L−1 in a laboratory scale. Furthermore, we demonstrate its binding with KD values in the lower nanomolar range (independently of Strep‐tag removal) and its capability to be blocked by serum antibodies in a competition ELISA with Strep‐Tactin‐HRP as a proof‐of‐concept. In addition, we assess the capacity of RBD to bind native dimeric ACE2 overexpressed in human cells and its antigen properties with specific serum antibodies. Finally, for completeness, we analyzed RBD microheterogeneity associated with glycosylation and negative charges, with negligible effect on binding either with antibodies or shACE2. Our system represents an accessible and reliable tool for designing in‐house surrogate virus neutralization tests (sVNTs), enabling the rapid characterization of neutralizing humoral responses elicited against vaccines or infection, especially in the absence of facilities to conduct virus neutralization tests. Moreover, our biophysical and biochemical characterization of RBD and shACE2 produced in S2 cells lays the groundwork for adapting to different variants of concern (VOCs) to study humoral responses elicited against different VOCs and vaccine formulations.

Characterization and application of recombinant Bovine Leukemia Virus Env protein

“…Sample preparation for MS analyses was achieved with soluble proteins or bands from Coomassie-stained SDS-PAGE as described before 46 .…”

“…Stably transfected Drosophila melanogaster S2 cells have appeared as a major platform for recombinant protein expression 43 , 44 . N-glycosylation in S2 insect cells gives oligomannosidic or paucimannosidic glycans with lower complexity than mammalian cells 45 , making it a suitable eukaryotic expression system for biophysical, biochemical and structural characterization of recombinant secreted viral glycoproteins, as well documented in Carrión et al and others 46 , 47 . Furthermore, S2 cells are currently being utilized for the production of potential vaccine antigens, demonstrating excellent performance in inducing neutralizing antibodies, as recently evidenced in the production of the Zika E viral protein 48 .…”