I am planning to clone an enzyme using the pET28a vector. I would like to ask whether the N-terminal His-tag alone would be sufficient for efficient expression and purification, or if it might cause the protein to misfold, making it difficult to purify.
Would it be more effective to place the His-tag at the C-terminal of the enzyme instead? In this case, I would add a stop codon in the reverse primer.
Which approach would be better for ensuring proper folding, expression, and successful purification of the enzyme?
I'd suggest trying both constructs, because you never know which will work out better.
The N-terminal His-tag on the pET28a vector is usually compatible for efficient expression and purification. Still, it could cause misfolding if the enzyme's N-terminus is important for correct folding or function. If this is a concern, place the His-tag at the C-terminus with a stop codon in the reverse primer may improve folding and activity.
pET28a has two His-tag sequences, so you can have a C-term His-tag without introducing one in the reverse primer. For a C-terminal His-tag you need to remove the N-terminal one by cloning with NcoI at the 5'-end of your insert.
I would also try both variants if you have no reason to assume that one might be problematic (some proteins require tag-free start or end region for dimerization etc.).
Do you know anything about the folded structure of the enzyme or important functional domains?
If yes, then put the tag on the end that is on the "outside" of the enzyme and not in a regulatory/binding site/other important functional part.
If not, then the best strategy is what everyone else has suggested - just make both versions and test them empirically.
Check the Alphafold predicted structure of your protein. If the N-terimus is deeply buried in the protein or involved in protein-protein interactions, try the C-terminus. Probably best to do both though since it isn't that much extra work.
If you added the Nco 1 cut site to the forward primer, you should not remove the stop codon because the his tag will be at the C terminal, if you placed the his tag at the N terminal, there will be no problem whether you remove the stop codons or not and expression and purification will occur.
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