Each protein is different, but there are indeed similarities within classes. It can be dangerous/unproductive to generalize -- you will find out experimentally whether you can assume things or not.
Depending on the expression method and the design of your construct you may or may not have a tag. Historically, a lot of eukaryotic protein kinases tend to be expressed in insect cells with a GST or His-GST tag. Protein kinases tend to favorably express in insect cells but the GST thing is in my experience more of a historical trend than a real requirement - I've expressed quite a few kinases with a simple His-tag. So, we can perhaps assume your first purification is a tag of some sort, and that you can cleave the GST away (His-tag typically does not have to be cleaved for crystallization, though it can certainly help). Depending on the purity and homogeneity of the enzyme you may want to try an orthogonal second step - a dye affinity resin or an ion exchanger, followed by size exclusion if necessary. If your kinase is autophosphorylated or if it's phosphorylated by another kinase you may have a mixed population of phosphates which can often be resolved by careful ion exchange chromatography (monoQ typically) either alone or in combination with phosphatase treatment. Note that phosphatases may not remove all phosphates from a mature folded protein - if you have phsophorylation and you *must* have a completely non-phosphorylated protein you may have to try co-expression with a suitable phosphatase. If your first purification step is 'generic' (like an AS cut or a crude ion exchange) nothing much changes for the following steps except you will have less pure starting material and therefore may have to do more total steps (and perhaps add another technique into the mix like HIC etc.). Good luck, Artem On Sat, Jan 1, 2011 at 1:03 AM, Neeraj Kapoor <nkap...@mail.rockefeller.edu>wrote: > Hi All, > A very happy new year to everyone. I am beginning to purify a novel > kinase and was wondering if there's a standard protocol / method that can be > utilized to begin purification of a kinase. I am new to the field of kinases > and would appreciate any help with respect to the precautions / pitfalls > while dealing with kinase purification. > > Thanks and have a great year ahead! > Neeraj >
