title: Use of the uteroglobin platform for the expression of a bivalent antibody against oncofetal fibronectin in Escherichia coli.
abstract: Escherichia coli is a robust, economic and rapid expression system for the production of recombinant therapeutic proteins. However, the expression in bacterial systems of complex molecules such as antibodies and fusion proteins is still affected by several drawbacks. We have previously described a procedure based on uteroglobin (UG) for the engineering of very soluble and stable polyvalent and polyspecific fusion proteins in mammalian cells (Ventura et al. 2009. J. Biol. Chem. 284∶26646-26654.) Here, we applied the UG platform to achieve the expression in E. coli of a bivalent human recombinant antibody (L19) toward the oncofetal fibronectin (B-FN), a pan-tumor target. Purified bacterial L19-UG was highly soluble, stable, and, in all molecules, the L19 moiety maintained its immunoreactivity. About 50-70% of the molecules were covalent homodimer, however after refolding with the redox couple reduced-glutathione/oxidized-glutathione (GSH/GSSG), 100% of molecules were covalent dimers. Mass spectrometry studies showed that the proteins produced by E. coli and mammalian cells have an identical molecular mass and that both proteins are not glycosylated. L19-UG from bacteria can be freeze-dried without any loss of protein and immunoreactivity. In vivo, in tumor-bearing mice, radio-iodinated L19-UG selectively accumulated in neoplastic tissues showing the same performance of L19-UG from mammalian cells. The UG-platform may represent a general procedure for production of various biological therapeutics in E. coli.
authors.affiliation: Laboratory of Oncology, G. Gaslini Institute, Genova, Italy.
authors.forename: Elisa
authors.initials: E
authors.lastname: Ventura
authors.affiliation: Department of Health Science, Nuclear Medicine, University of Genoa, IRCCS AOU San Martino - IST, Genova, Italy.
authors.forename: Mattia
authors.initials: M
authors.lastname: Riondato
authors.affiliation: Department of Health Science, Nuclear Medicine, University of Genoa, IRCCS AOU San Martino - IST, Genova, Italy.
authors.forename: Gianmario
authors.initials: G
authors.lastname: Sambuceti
authors.affiliation: Department of Hearth, Environmental and Life Science, Center for Excellence in Biomedical Research, Genova, Italy.
authors.forename: Annalisa
authors.initials: A
authors.lastname: Salis
authors.affiliation: Department of Experimental Medicine and Center for Excellence in Biomedical Research, Genova, Italy.
authors.forename: Gianluca
authors.initials: G
authors.lastname: Damonte
authors.affiliation: Sirius-biotech, c/o Advanced Biotechnology Center, Genova, Italy.
authors.forename: Cinzia
authors.initials: C
authors.lastname: Cordazzo
authors.affiliation: Protein Expression and Purification Core Facility, EMBL Heidelberg, Heidelberg, Germany.
authors.forename: Hüseyin
authors.initials: H
authors.lastname: Besir
authors.affiliation: Laboratory of Oncology, G. Gaslini Institute, Genova, Italy.
authors.forename: Vito
authors.initials: V
authors.lastname: Pistoia
authors.affiliation: Sirius-biotech, c/o Advanced Biotechnology Center, Genova, Italy.
authors.forename: Luciano
authors.initials: L
authors.lastname: Zardi
mesh_terms: D000818:Animals; D000906:Antibodies / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005353:Fibronectins / Q000276:immunology*; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D013058:Mass Spectrometry; D051379:Mice; D016513:Mice, SCID; D017510:Protein Folding; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D014598:Uteroglobin / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000906:Antibodies; D005353:Fibronectins; D011994:Recombinant Proteins; C121542:oncofetal fibronectin; D014598:Uteroglobin