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":

@Cynthia,
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.

Jürgen


......................
Jürgen Bosch
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
Office: +1-410-614-4742
Lab:      +1-410-614-4894
Fax:      +1-410-955-2926
http://lupo.jhsph.edu








Mark J van Raaij
Laboratorio M-4
Dpto de Estructura de Macromoléculas
Centro Nacional de Biotecnología - CSIC
c/Darwin 3, Campus Cantoblanco
28049 Madrid
tel. 91 585 4616
email: mjvanra...@cnb.csic.es

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