Citation
- Authors: Svilenov HL. et al.
- Year: 2021
- Journal: Nat Commun 12 6737
- Applications: in vitro / DNA / jetOPTIMUS
- Cell type: Expi293F
Description: Human embryonic kidney Fibroblast
Known as: Expi 293-F, Expi, HEK-293 Expi
Method
All proteins were produced by transient expression in Expi293™ cells (Thermo Fisher) grown in Expi293™ expression medium at 37 °C with 8% CO2.
The cells were transfected with 1 µg plasmid per 1 mL of cell suspension (2:1 LC/Fd plasmid ratio for the Fabs, 2:1 LC/HC ratio for full-length IgGs, 4:1 plasmid ratio for BG505 SOSIP.664/furin) using the ExpiFectamine™ 293 transfection kit or jetOPTIMUS® (Polyplus) following the manufacturers’ protocols.
When jetOPTIMUS® was used, 1 mM sodium propionate and 5 mM sodium valproate were added to the cells 16–20 h after transfection in an analogical way to the addition of the enhancers from the ExpiFectamine™ 293 kit.
The cell supernatants were collected by centrifugation and filtered six days after transfection.
Abstract
Antibodies bind antigens via flexible loops called complementarity-determining regions (CDRs). These are usually 6-20 residues long. However, some bovine antibodies have ultra-long CDRs comprising more than 50 residues organized in a stalk and a disulfide-rich knob. The design features of this structural unit and its influence on antibody stability remained enigmatic. Here, we show that the stalk length is critical for the folding and stability of antibodies with an ultra-long CDR and that the disulfide bonds in the knob do not contribute to stability; they are important for organizing the antigen-binding knob structure. The bovine ultra-long CDR can be integrated into human antibody scaffolds. Furthermore, mini-domains from de novo design can be reformatted as ultra-long CDRs to create unique antibody-based proteins neutralizing SARS-CoV-2 and the Alpha variant of concern with high efficiency. Our findings reveal basic design principles of antibody structure and open new avenues for protein engineering.