Polysaccharides as Precipitants in Protein Crystallization for X-Ray Diffraction Studies

A novel polysaccharide-based method for protein crystallization has been developed for industrial purposes. In this paper, we describe the application of this new method in crystal growth for X-ray diffraction structural studies of proteins. We have tested the crystallization of three commercially available proteinslysozyme, xylose isomerase, and xylanaseand of two research targetsthe amphiphilic protein hydrophobins HFBI and HFBIIon the polysaccharides alginate, chitosan, pectin, and dextrin, and enzymatic hydrolysates of alginate and pectin. With the use of polysaccharides, we managed to grow crystals of each protein. Crystals of lysozyme, xylose isomerase, and xylanase were suitable for X-ray diffraction studies on the basis of size, shape, and diffraction tests. Data were collected on xylanase crystals, and the structure was refined to ensure that the polysaccharide has no effect on structure determination. Both hydrophobins also yielded crystals suitable for X-ray analysis, once a suitable additive was used in combination with the polysaccharide. Thus, we conclude that this novel method is applicable also in protein structural studies. We suggest that the underlying phenomena in crystallization with polysaccharides is the formation of a polysaccharide−protein phase where the phase boundary acts as the initiation point for crystal formation.