Dear Colleges,
there is a heterodimer protein (small and large subunits), it is being translated from a single mRNA, presumably, a single protein is made and it is later digested by a protease! producing small and large subunit ( according to Uniprot, large subunit has signal peptide and propeptide, and the small subunit has a propeptide ). there are three interadisulphid bonds and there is an intermolecular disulphid bond between the last residue of the large subunit with the first residue of the small subunit. In fact the two subunts are bonds to each other with the interdisolphid bond.
I am trying to clone, express and purify this protein (in E.coli) as a heterodimer. But I am not sure how to design the expressing construct.
Thank you in advance,
Is it a eukaryotic protein? if so, it is almost impossible to get a functional dimeric protein as the post translational modifications do not happen as in the eukaryotic cells. First, you need a protease to cleave the large protein and next, an enzyme that can form the disulfide bond to stitch them together. I strongly suggest you go for a eukaryotic expression system. However, it may be possible to do it in E.coli, if you can coexpress the enzymes required for the post translational modifications. I hope this is helpful to you.
Goodluck
You can try expressing the each subunit in separate open reading frames in a single plasmid. However, I doubt their dimerization in E.coli. Instead, you can do Baculovirus expression system in SF21 cells.
Express and purify them separately. Later cross-link them. May be you will get lucky.
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