title: Enzymatic antibody modification by bacterial transglutaminase.
abstract: Enzymatic posttranslational modification of proteins permits more precise control over conjugation site than chemical modification of reactive amino acid side chains. Ideally, protein modification by an enzyme yields completely homogeneous conjugates with improved properties for research or therapeutic use. As an example, we here provide a protocol for bacterial transglutaminase (BTGase)-mediated conjugation of cadaverine-derivatized substrates to an IgG1, resulting in stable bond formation between glutamine 295 of the antibody heavy chain and the substrate. This procedure requires enzymatic removal of N-linked glycans from the antibody and yields a defined substrate/antibody ratio of 2:1. Alternatively, a mutant aglycosylated IgG1 variant may be generated by site-directed mutagenesis. The mutation introduces an additional glutamine and yields a substrate/antibody ratio of 4:1 after coupling. Finally, we describe an ESI-TOF mass spectrometry-based method to analyze the uniformity of the resulting conjugates. The presented approach allows the facile generation of homogeneous antibody conjugates and can be applied to any IgG1 and a wide range of cadaverine-derivatized substrates.
authors.affiliation: Center for Radiopharmaceutical Sciences, Paul Scherrer Institute, Villigen, Switzerland.
authors.forename: Patrick
authors.initials: P
authors.lastname: Dennler
authors.forename: Roger
authors.initials: R
authors.lastname: Schibli
authors.forename: Eliane
authors.initials: E
authors.lastname: Fischer
mesh_terms: D001710:Biotin / Q000378:metabolism; D002103:Cadaverine / Q000378:metabolism; D005417:Flavobacterium / Q000201:enzymology*; D006031:Glycosylation; D007074:Immunoglobulin G / Q000235:genetics / Q000378:metabolism*; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000378:metabolism; D013058:Mass Spectrometry; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D013302:Streptomyces / Q000201:enzymology*; D011503:Transglutaminases / Q000378:metabolism*
chemical_list: D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D001710:Biotin; D011503:Transglutaminases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D002103:Cadaverine