Expression, purification, crystallization, and preliminary X-ray analysis of recombinant human saposin B.

Abstract Saposin B (also known as cerebroside sulfate activator or CSAct) is a small non-enzymatic glycoprotein required for the breakdown of cerebroside sulfates (sulfatides) in lysosomes. Saposin B contains three intramolecular disulfide bridges, exists as a dimer and is remarkably heat, protease, and pH stable. We have expressed the protein in a thioredoxin reductase deficient strain of Escherichia coli and purified the protein by heat treatment, followed by ion-exchange, gel filtration, and hydrophobic interaction chromatographies. The protein is properly folded as judged by the observed disulfide bond topology, the hydrogen–deuterium exchange rate, and the level of stimulation of sulfatide hydrolysis by arylsulfatase A. Crystals of human saposin B were grown by vapor diffusion and diffract to a resolution of 2.2 A. Despite obtaining only merohedrally twinned P3 1 native crystals, an untwined seleomethionine-substituted crystal belonging to space group P3 1 21 was also grown. The three-dimensional structure of saposin B protein will provide insights into how this 79 amino acid protein is able to solubilize relatively large membrane-bound lipid ligands.