Highly Oriented Recombinant Glycosyltransferases: Site-Specific Immobilization of Unstable Membrane Proteins by Using Staphylococcus aureus Sortase A

Recombinant glycosyltransferases are potential biocatalysts for the construction of a compound library of oligosaccharides, glycosphingolipids, glycopeptides. and various art let artificial glycoconjugates on the basis of combined chemical and enzymatic synthetic procedures. The structurally defined glycan-related compound library is a key resource both ill the basic Studies of their functional roles in Various biological processes and ill the discovery research of new diagnostic biomarkers and therapeutic reagents. Therefore, it is clear that file Immobilization of extremely unstable membrane-bound glycosyltransferases Oil some suitable supporting materials should enhance the operational stability and activity of recombinant enzymes and makes facile separation Of products and recycling use of enzymes possible. Until now, however, it seems that no standardized protocol preventing a significant loss of enzyme activity is available due to the lack of a general method of site-selective anchoring between glycosyltransferases and scaffold materials through a stable covalent bond. Here we communicate it versatile and efficient method for the immobilization of recombinant glycosyltransferases onto commercially available solid supports by means of transpeptidase reaction by Staphylococcus aureus sortase A. This protocol allowed for the First time highly specific conjugation at the designated C-terminal signal peptide moiety of recombinant human beta 1,4-galactosyltranseferase or recombinant Helicobacter pylori alpha 1,3-fucosyltransferase with simple aliphatic amino groups displayed on the surface of solid materials. Site-specifically Immobilized enzymes exhibited the desired sugar transfer activity, all improved stability, and it practical reusability required for rapid and large-scale synthesis of glycoconjugates. Considering that most mammalian enzymes responsible for the posttranslational modifications, including the protein kinase family, its well its glycosyltransferases are unstable and highly oriented membrane proteins. the merit of four strategy based oil site-specific transpeptidation is evident because the reaction proceeds only ill all engineered C-terminus without any conformational influence around the active sites of both enzymes its well its heptad repeats of rH FucT required to maintain native secondary and quaternary structures during the dimerization on cell Surfaces.