E.Coli expressed Calmodulin doesn't work?

[Isaac Li]

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 <IxMEM5.FJJ at campus-news-reading.utoronto.ca>, Isaac Li <isaac.li at utoronto.ca> wrote:
> 
>>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?
> 
> 
> 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. 
> 
> 
>>My CaM extraction procedures:
>>1. express Calmodulin-CFP-GST fusion in DH5a, 37 degrees, HB 
>>broth+ampicillin for 24 hours.
> 
> 
> 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...  
>  
> 
>>2. I sonicated the cells in Tris buffer
>>3. I span down the cell debris, and take the lysate to check for CaM 
>>activity.
> 
> 
> CaM has very hundreds activities. What's your assay? 
> 
> DK