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.

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