Hi! I'm wondering if anyone else has had a situation where they are purifying an enzyme, and only one clone is active? The cloned gene is genetically identical to other clones, but only this one clone appears to be active (I haven't checked the genetics of the plasmid). The enzyme is catalytically slow, all negatives are clean, it's repeatable, and we've transfected this clone multiple times.
Could the plasmid have been mutated throughout the cloning process, enabling more effective expression of the protein? The amounts of protein appear to be similar by western blot (no matter what clone is expressed). Any thoughts on this matter would be greatly appreciated.
You can perform a DNA sequencing to check the presence of your insert DNA (& any mutation if present). Sometimes during the process of cloning, mutations may occur. However, you already have a clone that gives your desired protein. Prepare a stock from this clone & transform as & when required for protein purification.
I would definitively sequence all plasmids to check for differences as this finding could help you to better understand what is needed for the enzyme to function. I would not ignore this finding ans there must be a reason. I assume you use same qualities (amounts) for transfection of all plasmids and that was validated.
It certainly could have picked up a mutation and sequencing would confirm it. Depending upon the protein being made and the expression system, their may be a strong selective pressure for mutants that don't express and therefore grow better which would amplify the occurrence of otherwise rare mutants.
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