Would the removal of methionine and alanine from the N-terminus of the Influenza A H3N8 nucleoprotein affect its function or structure, considering the goal of leaving a serine residue to create an optimal TEV protease cleavage site? My objective is to purify the nucleoprotein for use as an antigen in developing ELISA kits for diagnostic purposes, and I want to ensure that this modification does not compromise the protein's integrity or its suitability for diagnostic applications.
My guess is no, for the following reasons:
Structure:
The N-terminal few residues don't seem to fold into any specific structure as they don't resolve in the various crystal structures of NP.
Fusing a large tag to the N-terminus (e.g. maltose binding protein and a factor Xa cleavage site; Elton et al 1999 JVI) worked just fine for purification and biochemical studies
We also got function in a minireplicon assay with an NP that we'd lopped the first 11 residues off (Elton et al 1999, virology). Though that was using a very old fashioned minireplicon system (it was last century!) and I suspect we mght not get quite the same result with a modern pol I-launched system.
Antigenicity:
The only time we've ever noticed any strain/sequence-dependent difference in NP antigenicity is with the commercial anti-NP clone AA5H, which doesn't react well with pdm09 NPs, or at least not all of them. We've never formally mapped the epitope using this, but I don't think it's N-terminal. This doesn't say that animals won't produce abs against the region you're interested in, but NP is very immunogenic so I doubt it would be a major confounder even if they did.
Only one way to find out for sure though :)
Cheers
Paul
Paul Digard
Thank you, Paul, for your prompt and helpful response. I truly appreciate your guidance and will definitely give it a try. Fingers crossed we manage to make it work!
- Badges
- Science method
- Similar topics
- Biological Science
- Ecology