Expression and purification of SARS-CoV-2 ectodomains were conducted as previously described26 (link). Briefly, constructs encoded the SARS-CoV-2 S ectodomain (residues 16-1206 of the early SARS-CoV-2 GenBank MN985325.1 sequence isolate with 2P35 (link) or 6P36 (link) stabilizing mutations, a mutated furin cleavage site between S1 and S2, a C-terminal TEV site, foldon trimerization motif, octa-His tag, and AviTag) were used to express soluble SARS-CoV-2 S ectodomains. Constructs encoding the SARS-CoV-2 RBD from GenBank MN985325.1 (residues 331-524 with C-terminal octa-His tag and AviTag) and mutant RBDs were made as described26 (link), SARS-CoV-2 2P S, 6P S, and RBD proteins were purified from the supernatants of transiently-transfected Expi293F cells (Gibco) by nickel affinity and size-exclusion chromatography26 (link). Peak fractions were identified by SDS-PAGE, and fractions corresponding to S trimers or monomeric RBDs were pooled and stored at 4°C. Fabs and IgGs were expressed, purified, and stored as described45 ,46 .
Protocol detail
SARS-CoV-2 Spike Protein Expression and Purification
Partial Protocol Preview
This section provides a glimpse into the protocol.
The remaining content is hidden due to licensing restrictions, but the full text is available at the following link:
Access Free Full Text.
Corresponding Organization :
Other organizations : California Institute of Technology, Rockefeller University
Top 5 similar protocols
Protocol cited in 31 other protocols
Variable analysis
independent variables
- Constructs encoding the SARS-CoV-2 S ectodomain (residues 16-1206) with 2P or 6P stabilizing mutations, a mutated furin cleavage site, a C-terminal TEV site, foldon trimerization motif, octa-His tag, and AviTag
- Constructs encoding the SARS-CoV-2 RBD (residues 331-524) with C-terminal octa-His tag and AviTag
- Mutant RBDs
- Expression and purification of SARS-CoV-2 ectodomains
- Purification of SARS-CoV-2 2P S, 6P S, and RBD proteins from the supernatants of transiently-transfected Expi293F cells
- Identification of peak fractions by SDS-PAGE
- Pooling of fractions corresponding to S trimers or monomeric RBDs
- Experimental conditions and procedures for expression and purification of SARS-CoV-2 ectodomains, as previously described in the referenced publication (link provided)
- No positive or negative controls are explicitly mentioned in the provided information
Annotations
Based on most similar protocols
Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.
Sign up for free or login to display all annotations
As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!