Use of the uteroglobin platform for the expression of a bivalent antibody against oncofetal fibronectin in Escherichia coli

Elisa Ventura, Mattia Riondato, Gianmario Sambuceti, Annalisa Salis, Gianluca Damonte, Cinzia Cordazzo, Hüseyin Besir, Vito Pistoia, Luciano Zardi

Research output: Contribution to journalArticle

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.

Original languageEnglish
Article numbere82878
JournalPLoS One
Volume8
Issue number12
DOIs
Publication statusPublished - Dec 19 2013

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Uteroglobin
fibronectins
Escherichia coli
antibodies
Antibodies
glutathione
proteins
Molecules
recombinant antibodies
Glutathione Disulfide
biopharmaceuticals
biological production
Cells
neoplasms
protein depletion
Proteins
cells
Tumors
radio
Bearings (structural)

ASJC Scopus subject areas

  • Agricultural and Biological Sciences(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Medicine(all)

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Use of the uteroglobin platform for the expression of a bivalent antibody against oncofetal fibronectin in Escherichia coli. / Ventura, Elisa; Riondato, Mattia; Sambuceti, Gianmario; Salis, Annalisa; Damonte, Gianluca; Cordazzo, Cinzia; Besir, Hüseyin; Pistoia, Vito; Zardi, Luciano.

In: PLoS One, Vol. 8, No. 12, e82878, 19.12.2013.

Research output: Contribution to journalArticle

Ventura, Elisa ; Riondato, Mattia ; Sambuceti, Gianmario ; Salis, Annalisa ; Damonte, Gianluca ; Cordazzo, Cinzia ; Besir, Hüseyin ; Pistoia, Vito ; Zardi, Luciano. / Use of the uteroglobin platform for the expression of a bivalent antibody against oncofetal fibronectin in Escherichia coli. In: PLoS One. 2013 ; Vol. 8, No. 12.
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