We once had a more-or-less MBP-sized fragment before cleavage, but
this turned to be a spontaneous mutation. This expression experiment
had been started from a glycerol stock with an unknown number of
growth cycles prior to expression.
Starting from a fresh transformation with the purified and sequenced
plasmid solved the problem.
Since then, I insist everybody does a fresh transformation before
every expression experiment and not generate extra growth/dilution
cycles beyond the normal transformation, growth on plate, overnight
culture, dilution into large-scale expression culture.
Quoting "Bosch, Juergen":
On Nov 4, 2012, at 10:58 AM, Cynthia Kinsland wrote:
Since you're seeing the MBP band, it sounds as if you're getting
some cleavage. If there were no cleave you would only see the
fusion and TEV bands on the gel.
I think that is a wrong assumption.
He did not specify if he sees the MBP band also just before TEV
addition - it might also be truncation products which we happen to
see all the time. The ratio varies depending on the construct but it
can be as bad as a 1:1 ratio. You can really only tell if TEV
cleaves if you do a time course experiment at RT with a defined
amount of your protein and see if the fusion construct decreases. An
alternative for the lack of your 17kDa desired band is simply your
fusion construct is cleaved but your cleaved product might a) not be
soluble at that pH or b) aggregates and precipitates.
You might be able to perform the cleavage on the Amylose column
keeping a constant flow cycling.
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Mark J van Raaij
Dpto de Estructura de Macromoléculas
Centro Nacional de Biotecnología - CSIC
c/Darwin 3, Campus Cantoblanco
tel. 91 585 4616