The gene for nanobody C1 (NbC1) and F2 (NbF2) and were codon-optimized using the IDT Codon Optimization Tool, synthesized as a ready-to-clone gene fragment (Integrated DNA Technologies), and cloned into the phagemid vector pADL-23c. The nanobodies were produced as previously described (Huo et al., 2021 (link)). Briefly, the plasmid was transformed into the WK6 E. coli strain and protein expression induced by 1 mM IPTG grown overnight at 28°C. Periplasmic extract was prepared by osmotic shock, and the nanobody protein was purified with a 5 mL HisTrap nickel column (Cytiva), followed by size exclusion with a Hiload 16/60 Superdex 75 column.
Nutalai R., Zhou D., Tuekprakhon A., Ginn H.M., Supasa P., Liu C., Huo J., Mentzer A.J., Duyvesteyn H.M., Dijokaite-Guraliuc A., Skelly D., Ritter T.G., Amini A., Bibi S., Adele S., Johnson S.A., Constantinides B., Webster H., Temperton N., Klenerman P., Barnes E., Dunachie S.J., Crook D., Pollard A.J., Lambe T., Goulder P., Paterson N.G., Williams M.A., Hall D.R., Mongkolsapaya J., Fry E.E., Dejnirattisai W., Ren J., Stuart D.I, & Screaton G.R. (2022). Potent cross-reactive antibodies following Omicron breakthrough in vaccinees. Cell, 185(12), 2116-2131.e18.
Other organizations :
Oxford University Hospitals NHS Trust, Medawar Building for Pathogen Research, Medway School of Pharmacy, University of Kent, Mahidol Oxford Tropical Medicine Research Unit
Codon optimization of the gene for nanobody C1 (NbC1) and F2 (NbF2) using the IDT Codon Optimization Tool
Transformation of the plasmid into the WK6 E. coli strain
Protein expression induced by 1 mM IPTG
Overnight growth at 28°C
dependent variables
Nanobody protein production
Purification of the nanobody protein using a HisTrap nickel column and size exclusion with a Hiload 16/60 Superdex 75 column
control variables
Osmotic shock to prepare the periplasmic extract
controls
No positive or negative controls were 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.
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
