title: Engineering of an anti-epidermal growth factor receptor antibody to single chain format and labeling by Sortase A-mediated protein ligation.
abstract: Sortase-mediated protein ligation is a biological covalent conjugation system developed from the enzymatic cell wall display mechanism found in Staphylococcus aureus. This three-component system requires: (i) purified Sortase A (SrtA) enzyme; (ii) a substrate containing the LPXTG peptide recognition sequence; and (iii) an oligo-glycine acceptor molecule. We describe cloning of the single-chain antibody sc528, which binds to the extracellular domain of the epidermal growth factor receptor (EGFR), from the parental monoclonal antibody and incorporation of a LPETGG tag sequence. Utilizing recombinant SrtA, we demonstrate successful incorporation of biotin from GGG-biotin onto sc528. EGFR is an important cancer target and is over-expressed in human tumor tissues and cancer lines, such as the A431 epithelial carcinoma cells. SrtA-biotinylated sc528 specifically bound EGFR expressed on A431 cells, but not negative control lines. Similarly, when sc528 was labeled with fluorescein we observed antigen-specific labeling. The ability to introduce functionality into recombinant antibodies in a controlled, site-specific manner has applications in experimental, diagnostic, and potentially clinical settings. For example, we demonstrate addition of all three reaction components in situ within a biosensor flow cell, resulting in oriented covalent capture and presentation of sc528, and determination of precise affinities for the antibody-receptor interaction.
authors.affiliation: CSIRO Materials Science and Engineering, 343 Royal Parade, Parkville, Victoria, 3052, Australia.
authors.forename: Mariusz P
authors.initials: MP
authors.lastname: Madej
authors.forename: Gregory
authors.initials: G
authors.lastname: Coia
authors.forename: Charlotte C
authors.initials: CC
authors.lastname: Williams
authors.forename: Joanne M
authors.initials: JM
authors.lastname: Caine
authors.forename: Lesley A
authors.initials: LA
authors.lastname: Pearce
authors.forename: Rebecca
authors.initials: R
authors.lastname: Attwood
authors.forename: Nick A
authors.initials: NA
authors.lastname: Bartone
authors.forename: Olan
authors.initials: O
authors.lastname: Dolezal
authors.forename: Rebecca M
authors.initials: RM
authors.lastname: Nisbet
authors.forename: Stewart D
authors.initials: SD
authors.lastname: Nuttall
authors.forename: Timothy E
authors.initials: TE
authors.lastname: Adams
mesh_terms: D019881:Aminoacyltransferases / Q000235:genetics / Q000378:metabolism*; D019138:Antibodies, Blocking / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001710:Biotin / Q000378:metabolism; D045744:Cell Line, Tumor; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D066246:ErbB Receptors / Q000037:antagonists & inhibitors* / Q000276:immunology; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D057127:Single-Chain Antibodies / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D013194:Staining and Labeling / Q000379:methods*
chemical_list: D019138:Antibodies, Blocking; D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; D001710:Biotin; D019881:Aminoacyltransferases; C120542:sortase A; D066246:ErbB Receptors; D003546:Cysteine Endopeptidases