title: In vitro sortagging of an antibody fab fragment: overcoming unproductive reactions of sortase with water and lysine side chains.
abstract: Sortase A from Staphylococcus aureus attracts growing interest for its use in biotechnological protein modification. This enzyme binds to a short signal sequence at the C terminus of a target protein, cleaves it by formation of an acyl-enzyme intermediate, and subsequently attaches an oligoglycine with a peptide bond. In this work, we explored its usability for the modification of the L19 Fab fragment (specific for fibronectin ED-B), a promising candidate for antibody-based cancer therapy. The Fab fragment was expressed with a sortase signal sequence attached to its light chain, and was successfully modified with a fluorescent oligoglycine probe in good yield. Our interest focused on performance under conditions of limited oligoglycine concentrations. Two unproductive side reactions of sortase were observed. The first was hydrolysis of the acyl-enzyme intermediate; in the second, sortase accepted the ε-amino group of lysine as substrate, thereby resulting in polypeptide crosslinking. In case of the L19 Fab fragment, it led to the covalent connection of the heavy and light chains. Both side reactions were effectively suppressed by sufficient concentrations of the oligoglycine probe.
authors.affiliation: Bayer Schering Pharma AG, Aprather Weg 18a, 42113 Wuppertal, Germany.
authors.forename: Sina
authors.initials: S
authors.lastname: Möhlmann
authors.forename: Christoph
authors.initials: C
authors.lastname: Mahlert
authors.forename: Simone
authors.initials: S
authors.lastname: Greven
authors.forename: Peter
authors.initials: P
authors.lastname: Scholz
authors.forename: Axel
authors.initials: A
authors.lastname: Harrenga
mesh_terms: D019881:Aminoacyltransferases / Q000737:chemistry* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry* / Q000378:metabolism; D008239:Lysine / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D010455:Peptides / Q000378:metabolism; D013379:Substrate Specificity; D014867:Water / Q000737:chemistry* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D007140:Immunoglobulin Fab Fragments; D010455:Peptides; D014867:Water; D019881:Aminoacyltransferases; C120542:sortase A; D003546:Cysteine Endopeptidases; D008239:Lysine