title: Site-specific modification of a single-chain antibody using a novel glyoxylyl-based labeling reagent.
abstract: A novel, highly specific protein modification approach is described. By using conventional molecular cloning techniques, a protein can be constructed and expressed such that the N-terminal residue is replaced by cysteine. Its 1,2-aminothiol structure reacts very specifically with a glyoxylyl group at pH 7 or below, forming a relatively stable thiazolidine bridge. Therefore, a glyoxylyl-based labeling agent (e.g., radioactive tags, fluorescent probes, biotin) can be used to specifically modify a protein at its N-terminus. To highlight this novel approach, a recombinant anti-insulin single chain antibody (scFv) was specifically biotinylated at its N-terminus even in the presence of other proteins in the total cell lysate. The glyoxylyl-biotinylated scFv retained binding activity similar to unmodified scFv.
authors.affiliation: Arizona Cancer Center, 1515 North Campbell, Tucson, Arizona 85724, USA.
authors.forename: Z G
authors.initials: ZG
authors.lastname: Zhao
authors.forename: J S
authors.initials: JS
authors.lastname: Im
authors.forename: K S
authors.initials: KS
authors.lastname: Lam
authors.forename: D F
authors.initials: DF
authors.lastname: Lake
mesh_terms: D000595:Amino Acid Sequence; D000915:Antibody Affinity; D001483:Base Sequence; D019921:Biotinylation; D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli; D006038:Glyoxylates / Q000737:chemistry*; D007128:Immunoglobulin Fragments / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D007202:Indicators and Reagents / Q000737:chemistry; D007328:Insulin / Q000276:immunology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology
chemical_list: D006038:Glyoxylates; D007128:Immunoglobulin Fragments; D007202:Indicators and Reagents; D007328:Insulin; D011994:Recombinant Proteins; D003545:Cysteine