Further studies on the site-specific protein modification by microbial transglutaminase

A guinea pig liver transglutaminase (G-TGase)-mediated procedure for the si te-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 req uirements as compared to the G-TGase, tends to catalyze an acyl transfer re action between the gamma -carboxamide group of a intact protein-bound gluta mine residue and various primary amines. To demonstrate the applicability o f the M-TGase-catalyzed protein modification in a drug delivery system, we have utilized recombinant human interleukin 2 (rhIL-2) as the target protei n 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. Pept ide mapping of (Gal)(3)-modified rhIL-2 ((Gal)(3)-rhIL-2) by liquid chromat ography-electrospray ionization mass spectrometry (LC-ESI/MS) suggested tha t 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-c atalyzed methodology for the preparation of protein conjugates for clinical use.