The protein expression is good. I get the expected protein size but it has no nitrilase activity. What's the problem?
This can indeed make problems. You are adding six positive charges to the end of the protein, which can severely interfere with folding or function. Did you try to express your nitrilase without a tag to see if it comes out active? When this is the case then the His tag is most likely the problem. If the untagged version is inactive as well, then it can be a problem with expression, folding etc.
Indeed, expressing the untagged protein would allow you to see whether the His tag is interfering with the folding. Note that the Pyrococcus furiosus nitrilase was expressed without tags, for structural determination. Another possibility is to re-clone the gene so that the His tag is fused at the other end of the polypeptide (if you have it at the N-terminus, move it to the C-terminus). Or change the tag altogether. Does your nitrilase require cofactors? They may be essential for folding, and E. coli may not be providing sufficient amounts. After purification, does the protein elute from size exclusion at the expected molecular weight, or do you have aggregation? Food for thought...
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