Human glycosylation enzymes: challenges and solutions for protein expression

Mammalian enzymes involved in glycan synthesis and catabolism include glycosyltransferases (GTs), glycoside hydrolases (GHs), and numerous glycan modifying enzymes (designated "other" on the left menubar). These enzymes are generally synthesized in the mammalian endoplasmic reticulum and modify glycan structures in lumenal compartments of the secretory pathway and endocytic compartments. They are mostly glycosylated proteins, with disulfide bonds and other post-translational modifications, and are commonly difficult to generate as functional recombinant forms in bacteria. This repository has been developed to provide access to expression constructs for production of GTs and GHs (and a limited set of glycan modifying enzymes) for expression in bacteria, insect cells (baculovirus), and mammalian cells. The goal is to allow the production of soluble forms of the enzymes as catalytic domains, when possible, for use in biochemical, enzymatic, and structural studies. Many of the constructs have been designed as truncated forms devoid of a transmembrane domain as fusion proteins to affinity tags or other larger fusion proteins to facilitate affinity purification (see Construct Design link in the left menubar). All coding regions have been captured as Gateway® entry constructs in pDONR221 vectors or equivalent donor constructs generated by gene synthesis and have been transferred to custom Gateway® destination vectors for expression in the corresponding recombinant hosts. All constructs are available from the PSI Materials Repository at DNASU (see links in final Gene Records).

Searching and navigation to the respective coding regions and expression constructs can be made through links at the left by alphabetical list or by CAZy family. See the TUTORIAL link at the left to learn how to navigate this site.