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.

脊椎动物糖蛋白和糖脂是在复杂的生物合成途径中合成的，主要位于分泌途径的膜隔室中。催化这些反应的酶具有极强的特异性，但由于与它们作为功能性产物的重组表达相关的挑战，很少有酶被广泛表征。我们使用模块化方法创建了一个表达载体库，该库编码所有已知的人类糖基转移酶、糖苷水解酶和磺基转移酶，以及其他聚糖修饰酶。然后，我们将酶表达为哺乳动物和昆虫细胞宿主中分泌的催化结构域融合蛋白，纯化和表征酶的子集，并确定了一种酶的结构，即唾液酸转移酶 ST6GalNAcII。许多酶在两种宿主中都以高产量和相似水平产生，但个别蛋白质表达水平差异很大。该表达载体库将成为重组酶生产的转化资源，广泛支持结构-功能研究并扩大这些酶在糖化学和糖生物学中的应用。