X-ray studies of recombinant anti-testosterone Fab fragments: the use of PEC 3350 in crystallization

Recombinant anti-testosterone wild-type Fab fragment and mutant Fab fragmen ts with high binding selectivity developed by protein engineering have been crystallized with and without ligands. Crystals of these Fab fragments wer e obtained by the vapour-diffusion technique at room temperature using solu tions 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-anti gen complexes and from two uncomplexed Fab fragments, with resolutions rang ing from 2.10 to 3.1 Angstrom. Crystallization conditions for Fab fragments were found by using modifications of the low ionic strength PEG 3350 serie s. Suitable concentrations of PEG 400, MPD and glycerol solutions for use a s cryoprotectants in PEG 3350 solutions have been determined. One useful ob servation was that PEG 3350 is able to work alone as a cryoprotectant. The screening protocol used requires a smaller amount of protein material to ac hieve auspicious pre-crystals than previously Results support the claim tha t PEG 3350 is more suitable for the crystallization of Fab fragments than h igher molecular weight PEGs.