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Derivatization of Antibody Fab Fragments: A Designer Enzyme for Native Protein Modification
Author(s) -
Liebscher Sandra,
Kornberger Petra,
Fink Gerhard,
TrostGross EvaMaria,
Höss Eva,
Skerra Arne,
Bordusa Frank
Publication year - 2014
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201400059
Subject(s) - chemistry , combinatorial chemistry , protein engineering , peptide , derivatization , enzyme , fluorophore , substrate (aquarium) , reagent , conjugate , biocatalysis , peptide bond , biochemistry , fluorescence , organic chemistry , biology , ecology , mathematical analysis , physics , high performance liquid chromatography , mathematics , ionic liquid , quantum mechanics , catalysis
Bioconjugates, such as antibody–drug conjugates, have gained recent attention because of their increasing use in therapeutic and diagnostic applications. Commonly used conjugation reactions based upon chemoselective reagents exhibit a number of drawbacks: most of these reactions lack regio‐ and stereospecificity, thus resulting in loss of protein functionality due to random modifications. Enzymes provide an obvious solution to this problem, but the intrinsic (natural) substrate specificities of existing enzymes pose severe limitations to the kind of modifications that can be introduced. Here we describe the application of the novel trypsin variant trypsiligase for site‐specific modification of the C terminus of a Fab antibody fragment via a stable peptide bond. The suitability of this designed biocatalyst was demonstrated by coupling the Her2‐specific Fab to artificial functionalities of either therapeutic (PEG) or diagnostic (fluorescein) relevance. In both cases we obtained homogeneously modified Fab products bearing the artificial functionality exclusively at the desired position.