Enzymatically catalyzed HES conjugation using microbial transglutaminase: Proof of feasibility.
Polymer-drug and polymer-protein conjugates are promising candidates for the delivery of therapeutic agents. PEGylation, using poly(ethylene glycol) for the conjugation, is now the gold standard in this field, and some PEGylated proteins have successfully reached the market. Hydroxyethyl starch (HES) is a water-soluble, biodegradable derivative of starch that is currently being investigated as a substitute for PEG. So far, only chemical methods have been suggested for HES conjugation; however, these may have detrimental effects on proteins. Here, we report an enzymatic method for HES conjugation using a recombinant microbial transglutaminase (rMTG). The latter catalyzes the acyl transfer between the gamma-carboxamide group of a glutaminyl residue (acyl donors) and a variety of primary amines (acyl acceptors), including the amino group of lysine. HES was modified with N-carbobenzyloxy glutaminyl glycine (Z-QG) and hexamethylene diamine (HMDA) to act as acyl donor and acyl acceptor, respectively. Using (1)H NMR, the degree of modification with Z-QG and HMDA was found to be 4.6 and 3.9 mol%, 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. Finally, the process described in this study is a simple, mild approach to produce fully biodegradable polymer-drug and polymer-protein conjugates.
Besheer
Ahmed
A
Department of Pharmaceutical Technology and Biopharmacy, Institute of Pharmacy, Martin Luther University Halle-Wittenberg, Wolfgang-Langenbeck-Str. 4, D-06120 Halle/Saale, Germany.
Hertel
Thomas C
TC
Kressler
Jörg
J
Mäder
Karsten
K
Pietzsch
Markus
M
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055162:Biocatalysis; D004926:Escherichia coli / Q000235:genetics; D005240:Feasibility Studies; D006639:Histidine / Q000737:chemistry; D006895:Hydroxyethyl Starch Derivatives / Q000737:chemistry*; D015394:Molecular Structure; D008970:Molecular Weight; D010952:Plasma Substitutes / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012995:Solubility; D013379:Substrate Specificity; D011503:Transglutaminases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D006895:Hydroxyethyl Starch Derivatives; D010952:Plasma Substitutes; D011994:Recombinant Proteins; D006639:Histidine; D011503:Transglutaminases