Expression and Purification: Tips and Tricks
How pure should the protein be for crystallization?
Everyone knows that protein must be "very pure" for crystallization. It is expected that no extra bands will be seen on an overloaded gel. While this stringent requirement may benefit you when growing "x-ray quality crystals" for the purposes of data collection, the protein sample used for first crystallization trials may be quite dirty. In fact, it is good to have some contamination, because it may promote crystallization. This is because the purpose of first crystallization trials is not to obtain perfect crystals (although that would be lovely) but rather find the leading conditions which can be further optimized.
In practice, it is a good idea to take your protein after the first purification step (such as metal affinity column if you have his-tag) and give it a go with crystallization screens. Protein crystals often take two-three weeks to appear and you can use this time to optimize your purification protocol. Of course, it is important to realize that in some situations only final purified product will crystallize, so if preliminary trials with "not-so-pure" protein failed, you should consider running the same screens second time with highly purified batch.
Too little protein to see in the gel ?
Try TCA precipitation - an old time classic!
- 100% (w/v) Trichloroacetic acid (dissolve 50g TCA into ~35mL water)
- 1. Add 1 volume TCA to 4 volumes of protein sample.
- 2. Incubate on ice for 10 minutes.
- 3. Spin at 14K rpm for 5 minutes.
- 4. Remove supernatant, leaving protein pellet intact.
- 5. Wash pellet with 200µL ice-cold acetone.
- 6. Spin at 14K rpm for 5 minutes and discard supernatant.
- 7. Repeat steps 4-6 for a total of 2 washes.
- 8. Dry pellet on a heat block at 95°C for 5-10 minutes to drive off acetone.
- 9. Add loading buffer to sample and boil before loading on gel.
To increase solubility, follow Golovanov et al. 2004. In short,
- buy L-Arg and L-Glu (i.e. don't use salts)
- put L-Arg powder into water, start dissolving it, then add L-Glu powder, stir; add water to adjust volume as required
- max. solubility is 1 M. The pH of a 1M stock solution will be around 6.4 or close to neutral
- add stock solution (50mM) to your protein