Purification of DNA-binding proteins

- Protein crystallography

Main steps:

   - Protein purification
     - Introduction
     - Step-by-Step
     - Common sense
       - Common sense strategy
       - Purification protocol
       - Purification of soluble protein
       - Purification of DNA-binding protein
       - Timing of purification
     - Protocols
     - Charts & Tables
     - Appendix
   - Crystallisation

Special:

- Programs for crystallography
- X-ray detectors

Basic tutorials:

   - Chemistry
   - Protein
   - Peptide
   - Amino Acids

Xtal community:

- CCP4BB

Protocols and tips in protein purification or How to purify protein in one day.

DNA-binding proteins

DNA-binding proteins as a rule is not tolerant to low salt conditions and can reversibly or sometimes irreversibly precipitate. Also when cells are disrupted the DNA-binding protein could binds to DNA. To release the protein free we can try to destroy DNA by DNAase and apply high salt concentration to dissociate the protein from the DNA. However, if protein is purified to be used with DNA DNAase treatment should be avoid and disruption should be performed in 50mM tris-HCl buffer with 0.5-1.0M NaCl. If DNAase treatment is applied cells should be suspended and disrupted in the buffer favourable for the DNAase activity, 50mM tris-HCl, pH 7.5 8.0, 4-5mM MgCl₂. 5µg/ml Dnase I should be added. It is not necessary to incubate homogenates with the DNAase, it works during sonication.

After cells is disrupted and before debris is spun down, add 1/10-1/5 of sample volume of 5M NaCl to bring salt concentration to 0.5-1.0M in the homogenate. Spin down debris as usual.

The next step should be considered to be Heparin-Sepharose (Agarose) chromatography. Most (sadly, not all) DNA-binding proteins have high affinity to heparin. Typically to bind such a protein on the Heparin matrix sample should contain not very high concentration of salt, typically 0.05-0.2M. The most convenient way to reduce salt in the sample is dilution. This way you reduce protein concentration and therefore the possibility for the protein to bind back to DNA. Elute protein from the column with 10-15CBV of NaCl from the starting concentration (0.05-0.2M) to 1.5-2M. Normally purification fold is very high and only one additional step is required to get 90+% purity.

Often gel filtration works fine. Alternatively, ion exchange chromatography could be applied. For some DNA-binding proteins Phospho-cellulose or Phospho-Cellufine (CHISSO Corp. Japan) was found to give good purification. The latter matrix is preferable as it is modern rigid matrix allows flow rate of 2ml/cm²min. Ammonium sulphate cut could be useful as a first step of purification if the TP precipitates by less than 2M of Ammonium sulphate.

Purification diagram

Disruption by sonication in buffer 50 mM tris-HCl pH 8.0,
4mM MgCl₂, (1mM DTT), 5µg/ml Dnase I (optional).

Add 5M NaCl to get 0.5 - 1M in final sample.

Spin down debris

Dilution with buffer 50mM tris-HCl pH 8.0 to appropriate salt concentration (0.1-0.2M)

Ammonium sulphate cut

Heparin-Sepharose Chromatography

Desalting by dialysis or fast gel filtration or dilution to appropriate salt concentration

Gel filtration

P-Cellufine Chromatography