Hampton Research

RAMC 1997 Round Table Notes

Organized by Bob Cudney and Allan D'Arcy
Location: Le Bischenberg, France

Production of glycoproteins with predefined glycosylation

During the RAMC 97 Meeting there was discussion about the production of glycoproteins with predefined glycosylation. Annie Hassell provided the following reference after the meeting: Davis, SJ, Puklavec, MJ, Ashford, DA, Harlos, K, Jones, EY, Stuart, DI, & Williams AF. Expression of soluble recombinant glycoproteins with predefined glycosylation: application to the crystallization of the T-cell glycoprotein CD-2. Protein Engineering 6: 229-232 (1993).

Filter the protein

People who prefer to filter the protein sample prior to crystallization experiments suggest trying one of the following: 300 kD, 0.02 micron, 0.1 micron, or 0.2 micron filters.

Looking for new additives to try?

Looking for new additives to try? Iodoacetic acid, iodoacetamide, and trifluoroethanol.

Polyamines

When using polyamines for RNA and DNA crystallization keep in mind that polyamines effect both sample solubility and crystal quality.

Agarase

Try using agarase to dissolve agarose gels when growing crystals in agarose gels.

Hydrophobic protein

When working with a hydrophobic protein add the detergent during the purification procedure. Also try adding sodium chloride along with the detergent during purification.

His tags

His tags were discussed again. It appears there is no consensus on whether to leave the tail on or chop it off. Everyone seemed to agree to leave the His tag one, try a screen and if no crystals are obtained, chop off the His tag and repeat the screen.

Another crystallization variable

Try a variety of buffers effective at the same pH when optimizing crystallization conditions since sometimes the buffer will bind the protein or the buffer may prevent a desired ligand from binding.

Cryo Tips

Experiencing and increase in mosaic spread? Try optimizing the cryoprotectant conditions, try optimizing the crystallization reagent, consider the speed of transfer, the speed of freezing, and the size of the crystal as variables.

Block the cryostream for a few seconds or even minutes to remove ice when you have poor cryo conditions.

Ice on crystal in cryostream? Poor liquid nitrogen onto crystal in cryostream.

Best temperature

What is the best temperature for sample storage? Try an ammonium sulfate precipitation and store at 4°C. When ready to set up sample, spin and resuspend in the crystallization buffer. Others suggest flash freezing in liquid nitrogen and then freezing the sample at -80°C.

Additive

Try beta-mercaptoethanol as a crystallization additive.

Cryoprotectant

Note that 50% v/v glycerol is required as a cryoprotectant in 0.2 M magnesium formate.

Different host cells

Try different host cells in a bauculovirus system to see different levels of glycosylation. Or use inhibitors to prevent glycosylation. Or remove the glycosylation for enzymes.