Expression, purification, crystallization, and preliminary X-ray analysis of recombinant human saposin B.
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 2.2A. 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.
Ahn
Victoria E
VE
Department of Medical Biophysics, University of Toronto, 610 University Avenue, Toronto, Ont., Canada M5G 2M9.
Faull
Kym F
KF
Whitelegge
Julian P
JP
Higginson
Jason
J
Fluharty
Arvan L
AL
Privé
Gilbert G
GG
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D003460:Crystallization; D004220:Disulfides; D004926:Escherichia coli; D015870:Gene Expression; D006023:Glycoproteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D049231:Saposins; D021241:Spectrometry, Mass, Electrospray Ionization; D049128:Sphingolipid Activator Proteins; D014961:X-Ray Diffraction
D004220:Disulfides; D006023:Glycoproteins; C483345:PSAP protein, human; D011994:Recombinant Proteins; D049231:Saposins; D049128:Sphingolipid Activator Proteins