X-ray studies of recombinant anti-testosterone Fab fragments: the use of PEG 3350 in crystallization.
Recombinant anti-testosterone wild-type Fab fragment and mutant Fab fragments with high binding selectivity developed by protein engineering have been crystallized with and without ligands. Crystals of these Fab fragments were obtained by the vapour-diffusion technique at room temperature using solutions of PEG 3350 with various biological buffers and with a wide pH range. So far, five data sets have been collected from crystals of three Fab-antigen complexes and from two uncomplexed Fab fragments, with resolutions ranging from 2.10 to 3.1 A. Crystallization conditions for Fab fragments were found by using modifications of the low ionic strength PEG 3350 series. Suitable concentrations of PEG 400, MPD and glycerol solutions for use as cryoprotectants in PEG 3350 solutions have been determined. One useful observation was that PEG 3350 is able to work alone as a cryoprotectant. The screening protocol used requires a smaller amount of protein material to achieve auspicious pre-crystals than previously. Results support the claim that PEG 3350 is more suitable for the crystallization of Fab fragments than higher molecular weight PEGs.
Valjakka
J
J
Department of Chemistry, University of Joensuu, 80101 Joensuu, Finland. valjakka@cc.joensuu.fi
Hemminki
A
A
Teerinen
T
T
Takkinen
K
K
Rouvinen
J
J
D002021:Buffers; D011232:Chemical Precipitation; D003080:Cold Temperature; D003460:Crystallization; D019314:Dehydroepiandrosterone Sulfate / Q000737:chemistry; D004058:Diffusion; D007128:Immunoglobulin Fragments / Q000737:chemistry*; D009994:Osmolar Concentration; D011092:Polyethylene Glycols / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D013739:Testosterone / Q000737:chemistry* / Q000276:immunology*; D014835:Volatilization
D002021:Buffers; D007128:Immunoglobulin Fragments; D011994:Recombinant Proteins; D011092:Polyethylene Glycols; D013739:Testosterone; D019314:Dehydroepiandrosterone Sulfate