About microscopic forms of life, including Bacteria, Archea, protozoans, algae and fungi. Topics relating to viruses, viroids and prions also belong here.
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Peruse the GE Healthcare Protein Purification site for useful information on protein purification by chromatography. I can’t say for sure, since I don’t know the specifics of the lactamase you’re trying to purify, but I don’t think Q-sepharose is the resin of choice for you. It depends on the pI of your protein. Penicillinase (bla) from E. coli has a theoretical pI of 9.4, according to ProtParam on the Expasy proteomics server. Q-type resins are anion exchangers (that is, they bind negatively charged species). To get lactamase (at least bla from E. coli) to be negatively charged, you need to use a pH greater than 9.4. In general, you want to avoid extremes of high or low pH unless you know your protein is stable under those conditions. Typical pHs for protein purification are in the range of (very roughly) 5.5 to 8.5.
I think you want to use an S-type resin (S Sepharose Fast Flow, for example) and do cation exchange at, say, pH 7 to 7.5. What pH and type of ion exchange resin to use depends on the pI of your specific lactamase, but bla from E. coli will be positively charged at pH 7 and should bind to Q Sephaose. After washing off the non-binding material, you then apply a salt gradient to elute the protein of interest. A typical first-time gradient is 0-1M NaCl applied over a 10-20 column volume; you can fine tune the gradient once you know where your protein elutes.
So the overall procedure would be:
1) grow the cells to produce the protein, and make a crude extract (I’m assuming you’re not using an affinity-tagged protein) in no or low salt buffer at pH 7-7.5. (If your protein is affinity-tagged, then the first step will be affinity chromatography, followed by ion exchange chromatography, and the procedure will have to modified slightly).
2) equilibrate the S Sepharose column at pH 7-7.5
3) apply the crude extract and wash with no/low salt buffer until the background absorbance at 280 nm is back to baseline indicating that all the non-binding proteins have been washed off the column
4) apply the salt gradient and collect fractions, pool those fractions containing your protein.
3 posts • Page 1 of 1
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