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
