[ccp4bb] kinase purification

2011-03-29 Thread Neeraj Kapoor 
Hi All,
 I am trying to express a kinase but unfortunately there is aggregation 
happening as the protein is purified over a column. SInce I am new to the field 
of kinase expression and purification, I was wondering if someone could provide 
me with a couple of good references that can hit the ground running for me. I 
would also very much appreciate any helpful suggestions that anyone might have.

thanks
Neeraj

Re: [ccp4bb] kinase purification

2011-03-29 Thread gauri misra 
You can use ATP agarose for purification and include the cofactor required
right from expression till purification steps.

On Tue, Mar 29, 2011 at 8:10 PM, Neeraj Kapoor nkap...@mail.rockefeller.edu
 wrote:

 Hi All,
 I am trying to express a kinase but unfortunately there is aggregation
 happening as the protein is purified over a column. SInce I am new to the
 field of kinase expression and purification, I was wondering if someone
 could provide me with a couple of good references that can hit the ground
 running for me. I would also very much appreciate any helpful suggestions
 that anyone might have.

 thanks
 Neeraj

Re: [ccp4bb] kinase purification

2011-03-29 Thread Kendall Nettles 
There is a paper from John Kuriyan on co-expressing a phosphatase with c-Src 
kinase domain to enable bacterial expression of homogenous protein (
PMID:
16260764). Also look at work from E. Goldsmith (PMID: 16829129)

Lastly, I would suggest general approaches, such as: varying the ends of the 
DNA construct; checking different buffers (EDTA, reducing agents, glycerol, pH, 
ligands) with use DLS or analytical gel filtration to check for aggregation. 
Also try low temp induction, or different fusion proteins.

Maybe this is a good thing. With our favorite protein we often get aggregation 
of half the protein. We assume this is the misfolded protein. We pellet this 
and have dozens of structures using the supernatant. So maybe your aggregation 
is a feature and not a bug.

Kendall Nettles


On Mar 29, 2011, at 8:10 PM, Neeraj Kapoor wrote:

Hi All,
I am trying to express a kinase but unfortunately there is aggregation 
happening as the protein is purified over a column. SInce I am new to the field 
of kinase expression and purification, I was wondering if someone could provide 
me with a couple of good references that can hit the ground running for me. I 
would also very much appreciate any helpful suggestions that anyone might have.

thanks
Neeraj