I have several monoclonal antibodies to Shiga toxin that I would love to convert to single chain variable fragments. I got the variable regions cloned and sequenced, but I don't quite know what to do next. Thank you!
Criag, you want to make and purify scFV protein? If so, what do you want to do with the scFVs (generally, not specifically)?
Adam
Thanks for replying, Adam. Yes, I want to construct, express, and purify the scFV's. Initially, they would be used for Shiga toxin immunoassays, ultimately I think they could be useful as therapeutics.
We have cloned some of mouse/human mAbs into the pFUSE vectors from InvivoGen. These contain a signal sequence and IgG Fc region from human, mouse, rabbit or rat (http://www.invivogen.com/pfuse-fc). The Fc is dual purpose for us: 1) purification and 2) detection. We then transfected suspension 293 cells (http://www.lifetechnologies.com/us/en/home/life-science/protein-expression-and-analysis/transfection-selection/freestyle--max-system.html) with the plasmid and purified scFV-Fc fusion protein from the supernatant with Protein A/G columns. That system worked well for us, though we haven't published the details. Other expression systems or different tags would work too, but we use the 293 Freestyle system for making all of the various recombinant mammalian proteins that we use in the lab. We used an (SGGGG)4 linker format to connect the VL and VH domains. Hope that helps.
If you don't mind me asking, why use 293 cells instead of bacteria to express the fusions? Is it because you don't want to have to deal with codon optimizing, or do they always go to inclusion bodies or something?
Two reasons really. 1) 293 cells are our standard system for making mammalian proteins. In the past, we had difficulty producing some glycoproteins in bacteria, so we moved to 293 cells and just stuck with them. 2) For the scFVs we actually tried bacteria as well as, but we did have difficulty extracting them from inclusion bodies and didn't pursue it further since 293 cells were working well.
Thank you so much for the advice, Adam. I think we'll try bacterial expression first since we don't have the cells or protocols for mammalian expression, and if it's not ideal we'll try mammalian cell culture like you.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Methods
- Cloning