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: [log in to unmask]
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