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

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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 γ-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.

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