Title : Expression system for structural and functional studies of human glycosylation
enzymes
Abstract :
- Vertebrate glycoproteins and glycolipids are synthesized in complex biosynthetic pathways localized predominantly within membrane compartments of the secretory pathway
- The enzymes that catalyze these reactions are exquisitely specific, yet few have been extensively characterized because of challenges associated with their recombinant expression as functional products
- We used a modular approach to create an expression vector library encoding all known human glycosyltransferases, glycoside hydrolases, and sulfotransferases, as well as other glycan-modifying enzymes
- We then expressed the enzymes as secreted catalytic domain fusion proteins in mammalian and insect cell hosts, purified and characterized a subset of the enzymes , and determined the structure of one enzyme , the sialyltransferase ST6GalNAcII
- Many enzymes were produced at high yields and at similar levels in both hosts, but individual protein expression levels varied widely
- This expression vector library will be a transformative resource for recombinant enzyme production, broadly enabling structure-function studies and expanding applications of these enzymes in glycochemistry and glycobiology