My current project involves use of an active o-methyltransferase enzyme protein, whose (his-tagged) expression on E. Coli was successfully induced with IPTG (1mM). The size of the expressed enzyme protein (both crude and pure) is in conformity with that of literatures, but lack the "o-methylation activity". What could lead to the lack of activity please?
Aminu, there could be a number of reasons, some of which are harder to prove than others. Firstly, is your enzyme cleaved properly prior to becoming active? This occurs with a lot with enzymes, they can be produced as a monomer which is inactive, and only becoming active upon cleavage to a disulphide-linked dimer. If so, can you show that this cleavage takes place? (e.g by running the protein on SDS-PAGE both before and after reduction). Secondly, is it possible that the protein doesn't adopt the correct conformation after extraction. This can sometimes happen if the extraction utilises high detergent concentrations, and then the product is put into conditions with very low detergent concentrations. Sometimes it is better to step down the detergent concentration more gradually if you can. Thirdly, the enzyme may be inappropriately glycosylated, especially if it is being produced in an organism it is not usually produced in. Checking this is more tricky. Fourthly, the his-tag may inhibit the correct active conformation of the enzyme. Can you remove the tag after purification of the enzyme? Hope this provides a few possibilities to test, good luck, Jon
It would help to have a positive control - a sample of authentic, active enzyme - to make sure your assay is set up properly and your substrates are OK. Check you SAM. It's not very stable, and the breakdown product S-adenosylhomocysteine, can act as a pretty potent inhibitor.
Thank-you Deakin and Adam.
SDS-PAGE of both lysate and purified enzyme showed that the enzyme is cleaved (as the buffers contain DTT and mercaptoethanol), the enzyme extraction procedures requires no detergent and the his-tagged enzyme was reported (by other researchers) to be active.
The enzyme activity assay is always run with both positive and negative controls; purified enzyme from bovine tissue and reactions without SAM respectively.
Thank-you once again for your response.
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