same protein produced in different cell lines

[Pow Joshi]

On 11/5/06, Samira <jeimysb At gmail.com> wrote:
> Hi Pow- Basically one of them (expressed in COS cells) is binding
> normally to my target protein (consistent with previous data), while
> the other shows reduced binding.

I can think of the tags beings a problem, especially if you have a
his-tag ...I have copied some papers here for your reference which
show that the his tag in a protein can lead to protein misfolding or
change in post translationals modifications (ergo, N-glycolsylation).

Also, I'd imagine that the amount of proteins produced in the two
transfections would be different ....I am not sure if you're doing
some kinect measurements or just Co-Ips with whole cell extracts.....
this is a bit of handwaving, however, I can imagine concentration
being affected if there's some sort of co-operativity.

The third suggection would be, if you think glycosylation is a
problems, like Wolfgang mentioned, you could check it out of the gel.

Hope this helps.

His tag issues:
 Halliwell, C., Morgan, G., Ou, C.-P. & Cass, A. (2001) Introduction
of a (poly) histidine tag in 1-lactate dehydrogenase produces a
mixture of active and inactive molecules. Anal Biochem. 295,
257–261.[Medline]
 Pekrun, K., Petry, H., Jentsch, K., Hunsmann, G. & Luke, W. (1995)
Reverse transcriptase enzyme from type 1 human immunodeficiency virus
using different baculoviral vector systems. Eur. J. Biochem. 234,
811–818.[Abstract]
 Kim, K., Yi, E., Baker, D. & Zhang, Y. (2001) Post-translational
modification of the N-terminal His tag interfers with the
crystallization of the wild-type and mutant SH3 domains from chicken
src tyrosine kinase. Acta Crystallogr. D57, 759–762

best,
pow
>
> On 11/4/06, Pow Joshi <pow.joshi At gmail.com> wrote:
> > On 3 Nov 2006 15:59:26 -0800, ointment <jeimysb At gmail.com> wrote:
> > > Hi all -
> > >
> > > I'm testing two versions of the same protein, given to me by two
> > > collaborators. One was produced in BHK cells (stable transfection), and
> > > the other in COS-7 cells (transient transfection). I was trying to test
> > > how these constructs bind to my target protein, and strangely enough,
> > > they are behaving differently in the binding assays.
> >
> > could you please specify how different is different?
> >
> > pow
> >
> >  Does anyone have
> > > insight on why this might be happening, i.e., differential
> > > glycosylation? Thanks!
> > >
> > > Samira
> > >
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> >
>