E.Coli expressed Calmodulin doesn't work?

Hi, My name is Isaac, I just entered the field of biology. I have been
trying to express human calmodulin in E.coli cells, and tried a binding
assay for its activities but it doesn't work, can anyone give me some
advices?

My CaM extraction procedures:
1. express Calmodulin-CFP-GST fusion in DH5a, 37 degrees, HB
broth+ampicillin for 24 hours.
2. I sonicated the cells in Tris buffer
3. I span down the cell debris, and take the lysate to check for CaM
activity.

What is the commonly used assay to test CaM binding to peptide? and how
do we usually get CaM working? I sorta recall that CaM folds really
well... so i don't think it would be a folding problem..
Thank you very much

Isaac

In article <[Only registered users see links. ]>, Isaac Li <[Only registered users see links. ]> wrote:

I can't give you an advice but I can assure you with 100% confidence
that calmodulin on its own expresses in E.coli very well and is perfectly
soluble and active.


Ah, so it's not calmodulin but Calmodulin-CFP-GST. Well, that's
entirely different thing! A priori, any fusion protein is NOT supposed
to have all properties of its components...


CaM has very hundreds activities. What's your assay?

DK

Hi DK, thanks for your reply.
I realize that fusion protein may be the problem, but it seems that CaM
in fusions are quite robust in many biosensor cases, the CaM-CFP-GST
construct binds to GST beads and expresses very well...
All I want is to verify that my CaM binds to M13 in vitro at this stage.
I have tried quite a few pull down assays, and none of them works...
e.g. I saturated Histag-CaM to Ni beads, and threw GFP-M13 into the
solution +Ca2+, there's no obvious pull down, but the GFP-M13 is
strongly pulled down by commercial CaM beads...

do you have any suggestions?
Thanks a lot!
Isaac

DK wrote:

In article <[Only registered users see links. ]>, Isaac Li <[Only registered users see links. ]> wrote:

Commercial CaM sepharose is made with recombinant protein,
so I do't know what the problem is.

DK

Thanks for your answer, if I just want to test the calmodulin function,
what should I do?

Isaac

In article <[Only registered users see links. ]>, Isaac Li <[Only registered users see links. ]> wrote:

Like I said, CaM has tons of functions and there are probably
subtle differences between species. Simplest: CaM+EDTA runs
differently on a SDS (!) gel gel than CaM + Ca2+. Since you have
a fusion, you might not be able to detect such shift.

If it's a real CaM, it must bind to phenyl-sepharose in strictly
Ca-dependent manner. CaM activates cAMP-dependent PDE -
this might be easy enough assay for you.