2 replies to this topic

[aodonnell311]

I've been expressing recombinant plant proteins in E. coli overnight (in PET vectors), extracting total protein using a commercial detergent, and assaying total protein extract with substrates that the protein should be able to methylate.

I've had very low activities and would like to improve them.  I've tried adding magnesium ions, adjusting the pH, and expressing, extracting, and assaying the proteins in cold environments.  I've also tried elevating the temperature of the reactions just above room temperature.

I think one mistake I made was attempting to extract the proteins on ice.  This may have resulted in a poor extraction.

Any suggestions?

Thanks!

[HOYAJM]

You could try purifying your enzymes with purification tags. Is there any specific reason you are using total protein extract? It is extra work, but optimization is much easier once you have purified enzymes.

If you are already using pET vectors, you could clone into the MCS and use their purification tags.

The temperature is important, but I think the most important part is the cell lysis. Proper cell lysis goes a long way in maximizing the activity and purity of an enzyme.

[aodonnell311]

Thanks very much Hoyajm, I will try purifying.  We are using total protein to save time and because it's the method used by lab whose results we'd like to repeat.