Dear colleagues,
I am currently working on the expression of two proteins encoded within the same operon. According to the literature, they seem to be expressed consecutively, suggesting coordinated regulation for their function in assembly and activity.
In my case:
- The first protein (HrcN) is a hexameric ATPase, with a molecular weight of ~50 kDa as a monomer (and ~300 kDa in its functional hexameric state).
- The second protein (HrcO) is monomeric, adopting a long helical structure, with a molecular weight of ~20 kDa.
Methodology I am using a plasmid construct that contains both genes. Only HrcN carries an N-terminal His6 tag (see attached scheme). Since both proteins are translated independently, my questions are:
I would be very interested to hear from anyone with experience in expressing proteins from the same operon, especially in cases where only one carries an affinity tag.
Thank you in advance for your suggestions and insights.
Do you know whether or not HrcO is a stable part of the hexameric molecule? If it is part of it, then presuming you are purifying native protein, I would think the stoichiometry would be preserved. But if it is only peripherally associated with the complex, or is just the next step in an enzymatic pathway, then it probably would not purify with the complex.
You could look at SDS page of crude extracts and see if both proteins can be seen on the gel.
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