Further Studies on the Site-Specific Protein Modification by Microbial Transglutaminase

Abstract

A guinea pig liver transglutaminase (G-TGase)-mediated procedure for the site-specific modification of chimeric proteins was recently reported. Here, an alternative method with advantages over the recent approach is described. This protocol utilizes a microbial transglutaminase (M-TGase) instead of the G-TGase as the catalyst. M-TGase, which has rather broad structural requirements as compared to the G-TGase, tends to catalyze an acyl transfer reaction between the gamma-carboxamide group of a intact protein-bound glutamine residue and various primary amines. To demonstrate the applicability of the M-TGase-catalyzed protein modification in a drug delivery system, we have utilized recombinant human interleukin 2 (rhIL-2) as the target protein and two synthetic alkylamine derivatives of poly(ethyleneglycol) (PEG12; MW 12 kDa) and galactose-terminated triantennary glycosides ((Gal)(3))) as the modifiers. For the M-TGase-catalyzed reaction with PEG12 and (Gal)(3), 1 mol of alkylamine was incorporated per mole of rhIL-2, respectively. Peptide mapping of (Gal)(3)-modified rhIL-2 ((Gal)(3)-rhIL-2) by liquid chromatography-electrospray ionization mass spectrometry (LC-ESI/MS) suggested that the Gln74 residue in rhIL-2 was site specifically modified with (Gal)(3). The PEG12-rhIL-2 and (Gal)(3)-rhIL-2 conjugates retained full bioactivity relative to the unmodified rhIL-2. In pharmacokinetic studies, PEG12-rhIL-2 was eliminated more slowly from the circulation than rhIL-2, whereas (Gal)(3)-rhIL-2 accumulated in the liver via hepatic asialoglycoprotein receptor binding. The results of this study expand the applicability of the TGase-catalyzed methodology for the preparation of protein conjugates for clinical use.

Conjugates between minor groove binders and Zn(II)-tach complexes: Synthesis, characterization, and interaction with plasmid DNA

“…Infrared spectra (IR) were recorded with a Nicolet Nexus 870 instrument operating in the ATR mode on a diamond crystal. All-cis-3,5-N,N-di-tert-butyloxycarbonyl-1-N-(4-aminobutyl)-1,3,5-triaminocyclohexane 8 (10), All-cis-3,5-N,N-di-tert-butyloxycarbonyl-1-N-(6-aminohexyl)-1,3,5-triaminocyclohexane 8 (11), All-cis-3,5-N,N-di-tert-butyloxycarbonyl-1-N-(8-aminooctyl)-1,3,5- triaminocyclohexane (12), 8 N-Benzyloxycarbonyl-2-bromo-ethylamine (15), 30 All-cis-3,5-N,N-di-tert-Butyloxycarbonyl-1,3,5-triaminocyclohexane (21, tach-DIBOC 8 ), All-cis-5-N-tert-Butyloxycarbonyl-1,3,5-triaminocyclohexane (28, tach-MONOBOC 8 ), 5-[((1H-Indol-2'-yl)carbonyl)amino]-lH-indole-2-carboxylic acid 28 (31), 5-[((Benzofuro-2'-yl)carbonyl)amino]-1H-indole-2-carboxylic acid 28 (32), were synthesized as reported.…”

“…8 Then, treatment of compounds 22e27, with hydrogen in the presence of PdeC afforded the desired amine derivatives 9e14. Compounds 1e7 were easily obtained as trifluoroacetate salts by coupling amino derivatives 9e14 with the bis-indole (31) or benzofuran-indole (32) carboxylic acids 28 in the presence of EDC at room temperature to afford amido compounds 33e39, followed by deprotection at the amino functions by treatment with TFA in CH 2 Cl 2 at room temperature.…”

Applications of glycosyltransferases in the site-specific conjugation of biomolecules and the development of a targeted drug delivery system and contrast agents for MRI

“…However, the heterogeneous nature of the glycan moiety on the recombinant glycoprotein causes poor coupling. Various investigators are developing chemoenzymatic methods for site-specific conjugation of biomolecules (18,19).…”

Transglutaminase-catalyzed covalent multimerization of camelidae anti-human TNF single domain antibodies improves neutralizing activity

“…These enzymes can be easily produced and have found several applications as biocatalysts in the food, cosmetic and textile industry (Griffin et al, 2002). Recently, MTG was used for biotinylation of monoclonal antibodies (Josten et al, 2000), and a poly(ethyleneglycol)-modification of human interleukin 2 (Sato et al, 2001). The ribonuclease S-tag-peptide was shown to work as a good peptidyl substrate in protein cross-linking reactions catalyzed by microbial transglutaminase (MTG) from S. mobaraensis exemplified with S-tagged enhanced green fluorescent protein (EGFP) as a model protein (Kamiya et al, 2003).…”