Site-Specific Modification of Interleukin-2 by the Combined Use of Genetic Engineering Techniques and Transglutaminase

Abstract

Exploring a new method for the site-specific incorporation of functional groups into proteins, we have studied the combined use of genetic engineering techniques and enzymatic methods. Specifically, a short peptide for use as a substrate of guinea pig liver transglutaminase (TGase) is introduced at the N terminus of human interleukin-2 (hIL-2). The expressed chimeric protein (rTG1-IL-2) is chemically modified at a glutamine site in the appended sequence by TGase-catalyzed transamination with two amines, monodansylcadaverine (MDC), or a constructed derivative of poly (oxyethylene) (POE3). For the TGase-catalyzed modifications with MDC and POE3, 1 mol of donor was incorporated per mole of rTG1-IL-2, respectively. N-Terminal sequence analysis of MDC-modified rTG1-IL-2 (MDC-rTG1-IL-2) showed that the Gln-4 residue in the chimeric protein was site specifically modified with MDC. On the other hand, tryptic mapping of POE3-modified rTG1-IL-2 (POE3-rTG1-IL-2) by matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOFMS) suggested that one of the Gln sites in the appended sequence was modified with POE3. The POE3-rTG1-IL-2 retained full bioactivity relative to the unmodified molecule and rhIL-2. This methodology could be a new and general route for the site-specific modification of proteins.

Enzymatic formation of modular cell-instructive fibrin analogs for tissue engineering

“…Several groups, including ours, have previously demonstrated that this enzymatic reaction can be exploited for a variety of applications in drug delivery and tissue engineering. For example, research groups have reported on the transglutaminase-catalyzed site-specific modification of proteins with PEG (PEGylation) [17,18], the cross-linking of PEG [19,20] or proteins such as collagen [21] or gelatin [22] into hydrogel networks, and also the specific covalent immobilization of exogenous bioactive ligands into fibrin networks to change its biomolecular characteristics and create celland tissue-specific microenvironments [23,24]. In the latter case, the release of covalently bound biomolecules is coupled to the proteolytic degradation of the fibrin gel associated with invading cells during remodeling processes, making this matrix platform attractive for diverse tissue regeneration applications [25,26].…”

Biochemical and biophysical characterization of lysozyme modified by PEGylation

“…Numerous approaches have been explored to deal with the problem of biological activity losses as a result of PEGylation: sitespecific PEGylation, sometimes in combination with site-specific mutagenesis (Goodson and Katre, 1990;Kinstler et al, 1996;Gaertner and Offord, 1996;Sato et al, 1996;Cazalis et al, 2004); protection of receptor-binding site during conjugation (Caliceti et al, 1993(Caliceti et al, , 1994; and reversible PEGylation (Peleg-Shulman et al, 2004;Zalipsky et al, 2007). Each of these approaches has its merits and potential effectiveness in specific situations, although all of them have nontrivial technical obstacles.…”

Protein-based tumor molecular imaging probes

“…gpTGase has been demonstrated in many cases to label various proteins fused with ‘Q-tag’, a 6- or 7-mer transglutaminase recognition sequence. Interleukin-2 and glutathione S -transferase have been attached with monodansyl cadaverine (Sato et al 1996) and fluorescein cadaverine (Taki et al 2004), respectively, by gpTGase. Lin and Ting (2006) demonstrated the labeling of Q-tagged proteins by gpTGase both in vitro and on the surface of living mammalian cells with biotin, fluorophores, and a benzophenone photoaffinity probe.…”