The Sulfs: Expression, Purification, and Substrate Specificity
Sulf-1 and Sulf-2 are endo-acting extracellular sulfatases. The Sulfs liberate 6-O sulfate groups, mainly from N, 6-O, and 2-O trisulfated disaccharides of heparan sulfate (HS)/heparin chains. The Sulfs have been shown to modulate the interaction of a number of protein ligands including growth factors and morphogens with HS/heparin and thus regulate the signaling of these ligands. They also play important roles in development and are dysregulated in many cancers. The establishment of the expression of the Sulfs and methods of assaying them has been desirable to investigate these enzymes. In this chapter, methods to express and purify recombinant Sulfs and to analyze HS structures in an extracellular fraction of HSulf-transfected HEK293 cells are described. The application of these enzymes for ex vivo degradation of an anti-HS epitope accumulated in the brain of a neurodegenerative disease model mouse is also described.
Key wordsHeparan sulfate Sulf-1 Sulf-2 Sulfatase Endo-acting enzyme Protein ligands Immunoblot Transfection FLAG-tag His-tag
Supported by Japanese Health and Labour Sciences Research Grants [H19-001 and H22-007], Grants-in-Aid from the Ministry of Education, Science, Sports and Culture [22790303 and 24590349, and Scientific Research on Innovative Areas] and in part by the Takeda Science Foundation.
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