title: Enzymatic labeling of a single chain variable fragment of an antibody with alkaline phosphatase by microbial transglutaminase.
abstract: Functional cross-linking of a single chain Fv fragment of anti-hen egg-white lysozyme antibody (scFv) and alkaline phosphatase (AP) was explored using microbial transglutaminase (MTG) from Streptomyces mobaraensis. A specific peptidyl linker for MTG was genetically fused to the N-terminus of each protein and the resultant proteins were obtained separately by bacterial expression. The recombinant peptide-tagged scFv and AP were site-specifically cross-linked by MTG through the extra peptidyl linkers in vitro, which mainly yielded the heterodimer (i.e., scFv-AP conjugate). The enzymatic cross-linking reaction had little influence on either the antigen-binding ability of the scFv moiety or the enzymatic activity of the AP moiety of the conjugate, allowing use within an enzyme-linked immunosorbent assay. The results obtained suggest that the enzymatic approach with MTG facilitates the posttranslational construction of functional fusion proteins.
authors.affiliation: Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Takazawa
authors.forename: Noriho
authors.initials: N
authors.lastname: Kamiya
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Ueda
authors.forename: Teruyuki
authors.initials: T
authors.lastname: Nagamune
mesh_terms: D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000737:chemistry / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D013302:Streptomyces / Q000201:enzymology / Q000235:genetics; D011503:Transglutaminases / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins; D011503:Transglutaminases; D000469:Alkaline Phosphatase