Enzymatically Catalyzed Conjugation of a Biodegradable Polymer to Proteins and Small Molecules Using Microbial Transglutaminase

Abstract

Hydroxyethyl starch (HES) is a water-soluble, biodegradable derivative of starch that is widely used in biomedicine as a plasma volume expander. Due to its favorable properties, HES is currently being investigated at the industrial and academic levels as a biodegradable polymer substitute for polyethylene glycol. To date, only chemical methods have been suggested for HESylation; unfortunately, however, these may have negative effects on protein stability. To address this issue, we have developed an enzymatic method for protein HESylation using recombinant microbial transglutaminase (rMTG). rMTG enzyme is able to catalyze the replacement of the amide ammonia at the γ-position in glutamine residues (acyl donors) with a variety of primary amines (acyl acceptors), including the amino group of lysine (Lys). To convert HES into a suitable substrate for rMTG, the polymer was derivatized with either N-carbobenzyloxy glutaminyl glycine (Z-QG) or hexamethylenediamine to act as an acyl donor or acyl acceptor, respectively. Using SDS-PAGE, it was possible to show that the modified HES successfully coupled to test compounds, proving that it is accepted as a substrate by rMTG. Overall, the enzymatic approach described in this chapter provides a facile route to produce biodegradable polymer–drug and polymer–protein conjugates under relatively mild reaction conditions.