Folding - (Jan/25/2011 )

Suppose we are trying expressing fusion protein of MBP-protein X in E coli. The construct used is pMAL with IPTG induction etc...

And suppose everything was successful: The vector has 0 mutation, the vector transformed successfully, the bacteria grew successfully, the amylose prep was successful, after buffer exchange in desired buffer, SDS gel showed you get a great band of your protein X that has the right kDa size and no truncation, no contamination. Life is good.

But how do you know that the protein has the correct folding that it will do its task properly?

Or should we just test if it is doing whatever it's supposed to do = it has correct folding?

My concern is that if it has different folding then maybe it has some other activities and probably different function as well, which may yield false positives/negatives, because SDS does not test folding.

RP-HPLC usually distinguishes between folded partially folded and unfolded proteins!!!
and yeah the best way is to check the activity...

Hola, as Prep says activity, HPLC, gel fitration are ways to know well folded and homogeneity of your pure protein, native gel could be another way, but i´m going tell you a case that happened to me. Purifiying bacterial produced GM-CSF which has 2 intramolecular disulfide bridges, in the elution of ion echanger colum, it appeared two peaks at 0.2 and 0.8 M NaCl both with the protein. analizing samples by PAGE with loading buffer without reducer and not boiled, 0.2M bands appeared as double band, that adding reducer converts in one, and 0.8M showed without reducer multimeric forms from intermolecular(bad folded) disulfide bridges. I don´t know how your protein is , and if it has intramolecular disulfides but in my case only the upper band of 0.2M NaCl seemed to be the well folded one, which total reduction transformed in the lower band. Buena suerte