Summary and new questions Re: [ccp4bb]: protein expression

[capricy gao]

Hi,

 

Thank you all so much for the responses and here are the summary for possible causes proposed from all the emails:

1) Sample precipitation without moving along the the gel

2) multiple oligomeric states of the protein

3) post-translational modification like Kinase

 

Basically protein looks smear on gel, so

 

More questions:

1) what are possible reasons for a protein soluble (not membrane protein) in TrisHCl (pH8.0) precipitates in isoelectric focusing gel? IEF gel is immobilized pH gradient from Amersham

2) Does original buffer where protein is sitting in have effect on IEF gel?

3) How oligomeric state affects the moving since IEF gel running has Urea and DTT in buffer?

4) What are the possible post-translational modifications for gene _expression_ in E coli?

 

&nb! sp;

Many thanks again,

 

Capricy

"John A. Newitt" <[EMAIL PROTECTED]> wrote:

At 8:14 AM -0800 1/5/06, capricy gao wrote:

>An eukaryotic gene was expressed in E coli cell. The protein is
>soluble. But it doesn't focus on isoelectric focusing gel. Protein
>sequencing shows that the polypeptide chain is intact, that is,
>no proteolysis is detected. Anybody met the similar problem?

If the eukaryotic protein is a kinase, heterogeneous
hyper-phosphorylation may explain your observation. I have seen this
for some eukaryotic kinases that were over expressed in E. coli.
Sometimes co-_expression_ with a phosphatase (e.g . Lambda PPase) or
phosphatase treatment of the purified protein in vitro will remove
much or all of the charge heterogeneity.
- John

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