Site-specific modification of interleukin-2 by the combined use of genetic engineering techniques and transglutaminase.
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.
Sato
H
H
Drug Delivery System Institute, Ltd., Science University of Tokyo, Chiba, Japan.
Ikeda
M
M
Suzuki
K
K
Hirayama
K
K
D000595:Amino Acid Sequence; D000818:Animals; D002103:Cadaverine / Q000031:analogs & derivatives / Q000378:metabolism; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D005973:Glutamine / Q000737:chemistry; D006168:Guinea Pigs; D006801:Humans; D007376:Interleukin-2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping; D010455:Peptides / Q000737:chemistry; D011092:Polyethylene Glycols / Q000378:metabolism; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017421:Sequence Analysis; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D011503:Transglutaminases / Q000378:metabolism*
D007376:Interleukin-2; D010455:Peptides; D011993:Recombinant Fusion Proteins; D005973:Glutamine; D011092:Polyethylene Glycols; D011503:Transglutaminases; C008542:monodansylcadaverine; D002103:Cadaverine