Recombinant expression and characterization of ligand binding to mutants ff 3-O-sulphotransferase-1

Heparan Sulphate (HS) is a highly charged glycosyaminoglycan (GAG) that is closely related to Heparin, a related GAG with a higher degree of sulphation. Heparin is used in the development of anticoagulant drugs. Currently, there is a lack of useful reagents for studying and distinguishing between complex GAGs like heparin/heparan sulphate from other GAGs. As a result, GAGs are often contaminated with other GAGs like chondroitin sulphate upon isolation. To improve GAG characterization, we aim to engineer a heparin reagent through the conversion of the HS modifying enzyme 3-O-sulphotransferase isoform 1 (3-OST-1). 3-OST-1 transfers a sulphate group to the 3-OH position of an N-sulpho-glucosamine linked to a glucuronic acid. This modification is essential for heparin used in the development of anticoagulant drugs like Arixtra. Four 3-OST-1 mutants, E90Q, E90K, E90H and E90R were generated using site-directed mutagenesis. The binding affinity for 3-OST-1 modified heparin was measured by surface plasmon resonance using the anticoagulant drug Arixtra. The effect of side chain and charge at position 90 among the four mutants in binding to Arixtra was investigated. The mutant with the greatest binding affinity was determined. Here in is the report on the expression, purification and binding analysis of the mutants to date.

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