we've had problems with Xa cleavage of MBP-fusions, so apols for asking, but are you sure cleavage took place? And if you are, have you tried, for example, high/low salt buffers with GPC (or even amylose) to reduce the binding?

Happy Xmas!

G

Sorry for the delayed response.

Yes we confirmed cleavage by SDS page. I have not tried high salt on the size exclusion column, nor amylose. Will consider that. Thank you!

I just reread your question. What about pMALC2 or similar? That works in our hands, but post Xa cleavage (to a native protein: no additional AA) we've had aggregation problems with some proteins (see below). Also pQE30Xa (hexahis tag) only adds a handful of AA upstream of the Xa site and that has been acceptable without cleavage for crystallography papers.

I recently discovered (not enough reading!!) that Xa is a heterodimer linked by SH groups. Some of our proteins really need DTT or similar to prevent aggregation. Hence our problems, I think!

Thanks again Geoff. pQE30Xa is a good thought. We are also working on a re-engineered form of pMAL-c5X that allows easy swapping of the affinity tag by either Gibson or restriction enzyme cloning methods.

Eric, in worst case, have the cDNA corresponding to the Xa-site and the GST tag custom synthesized and add it to your construct / replace the MBP tag.