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Question

    I am trying to purify a histidine tag Lim-homeo domain
(transcription factor) through a NTA-Ni column. After I had purified the
protein, I will analyse it through the light scattering machine and also
gel filtration but I always get aggregates. I thought it could be due to
the presence of contaminating DNA or maybe some problems with the
buffer. I have modified the buffer to include DNase (to get rid of the
DNA), 0.2M NaCl, 10% glycerol and using Tris as a buffering system. I
had got very low DNA contamination this time but the protein still
aggregates. Does anyone have any experience in trying to solubilise
similar proteins?

Summary of answers/suggestions

Although this is not a problem of 'Crystallographic Computing', in this form, the information could come in very handy.

1. Sometimes aggregation could be due to oxidation (in artificial environment) of cysteine residues leading to formation of disulphide bridges. Try using 0.1% beta-mercaptoethanol to prevent oxidation.
2. Use higher salt (0.5M) and glycerol to stabilise proteins.
3. Monitor the pH and keep it away from the pK of the protein.
4. When his-tag protein is purified through Ni-NTA column, during elution Ni often gets eluted along with the protein and binding of proteins to the eluted Ni could be the cause of aggregates. Try dialysing the protein against 10mM EDTA or just by addition of EDTA to the tubes used to collect fractions.
5. Combat the problems of aggregation by using organic solvent e.g. 6% methanol or ethanol. It is also found that tritonX or ACN can dissolve the aggregates. Using detergent screen from Hampton and then monitoring through DLS could be a good way of screening.
6. Try and cut the His-tag off. It is observed that solubility of His-tagged proteins can be influenced by the presence of the tag.
7. Add the target DNA to the transcription factor; the source of aggregation might be due to poorly folded protein.
8. Use an alternative column for purification of His-tag protein: Cobalt-TALON. Ions on the column will not detach because it has one more dental holding the ion.
9. It is possible to still use EDTA (to get rid of the Ni) even if a protein is a zinc binding protein. Try adding 1.5mM EDTA and 2mM zinc chloride.
10. Try using low pH to elute His-tag protein from the Ni-NTA column. It is also noted that a longer His-tag reduces protein solubility and favours aggregation.
11. It has been observed that b-mercaptoethanol can bind to the cysteine residues and form a covalent adduct. Try using other alternatives e.g. DTT.