Hi Neeraj, A few points to add to Artem's excellent advice:
If you would like to purify an untagged construct of your kinase, you could try an ATP affinity column. The caveats to this are 1.) the ATP resins can be expensive, and therefore it may be impractical to make a column with a high binding capacity, and 2.) Lots of proteins bind ATP, so your fractions might not be so clean (preceding your ATP column with an AS precipitation could help). Artem mentioned that often eukaryotic kinases are expressed in insect cells, and also touched on the fact that they can be phosphorylated by other kinases within your expression system. It may be worth a try to see if you can get your protein to express in a bacterial system, because most bacteria (notably laboratory strains of E.coli) lack S/T/Y kinases. Autophosphorylation, however, cannot really be avoided. Certain competent E.coli are pretty good for expressing eukaryotic proteins. You might try the BL21 CodonPlus Rosetta strain (available commercially - novagen I think, or maybe Stratagene) because it has a plasmid that expresses a number of tRNAs that correspond to RILP codons that are common in eukaryotes, but rare in E.coli. Good luck, Mike Thompson ----- Original Message ----- From: "Artem Evdokimov" <artem.evdoki...@gmail.com> To: CCP4BB@JISCMAIL.AC.UK Sent: Saturday, January 1, 2011 6:56:03 AM GMT -08:00 US/Canada Pacific Subject: Re: [ccp4bb] help with kinase purification 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 -- Michael C. Thompson Graduate Student Biochemistry & Molecular Biology Division Department of Chemistry & Biochemistry University of California, Los Angeles mi...@chem.ucla.edu
