I am using computational methods to design proteins that fold into specific shape. However, I found that, in reality, proteins after synthesis usually have tags (eg His tag) and liners, which could potentially affect the folding of my protein. Is there a way to synthesize a protein without any extra residues?
You need to have a cleavage site directly upstream of the desired sequence and downstream of the affinity tag. You can use a a cleavage site for TEV protease, for example, and cleave after purification. Alternatively, a methionine residue directly upstream of the desired sequence will allow you to cleave off the affinity tag using cyanogen bromide. You can't have any other met residues in the protein. The conditions are pretty harsh and the protein will need to be refolded. Self-splicing inteins can also be employed, but these may not be completely traceless.
Just synthesise the protein simply without any tag if it's possible, there are plenty of vectors for tagless protein production.
But if you'll include some tag with the cleaving site, you may affect the folding although you cleave it off afterwards.
You might be able to use his-tagged TEV and remove the cleaved his-tag from your protein as well as the protease by running it through a Ni-NTA column.
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Biochemical Techniques