title: Application of an alkaline phosphatase fusion protein system suitable for efficient screening and production of Fab-enzyme conjugates in Escherichia coli.
abstract: We report a novel vector system suitable for the efficient preparation of alkaline phosphatase (PhoA)-labelled antibody Fab fragments in Escherichia coli. The previously described pGE20 vector used for the functional expression of truncated heavy (Fd) and light (L) chains of Fab into the bacterial culture medium was modified by inserting the E. coli PhoA coding region 3' to the Fd cloning sites. The secreted Fd-PhoA fusions and L proteins were found to be disulfide linked and Fab-PhoA complexes, prepared with IgG1 antibodies recognizing specifically human tumor necrosis factor alpha, were shown to be useful for the rapid detection of antigen. When an additional short peptide was interposed between the Fd and PhoA domains, nearly all of the recombinant Fab-PhoA conjugates present in the culture supernatant retained both antigen binding and enzymatic activity. A method for the detection and selection of bacterial colonies expressing bifunctional hybrid molecules of desired antigen specificity was also developed. Taken together, the systems described permit the generation and production of Fab-PhoA conjugates in E. coli, which can replace conventionally prepared PhoA-labelled antibodies in appropriate immunoassays.
authors.affiliation: Ecole Supérieure de Biotechnologie de Strasbourg, France.
authors.forename: E
authors.initials: E
authors.lastname: Weiss
authors.forename: G
authors.initials: G
authors.lastname: Orfanoudakis
mesh_terms: D000469:Alkaline Phosphatase / Q000096:biosynthesis / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D018033:Antibodies, Bispecific / Q000096:biosynthesis / Q000235:genetics; D000937:Antigen-Antibody Reactions; D001483:Base Sequence; D001709:Biotechnology; D003001:Cloning, Molecular; D004274:DNA, Recombinant / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors; D006801:Humans; D007118:Immunoassay; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology
chemical_list: D018033:Antibodies, Bispecific; D004274:DNA, Recombinant; D007140:Immunoglobulin Fab Fragments; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; D000469:Alkaline Phosphatase