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: Elisa Ventura, Mattia Riondato, Gianmario Sambuceti, Annalisa Salis, Gianluca Damonte, Cinzia Cordazzo, Hüseyin Besir, Vito Pistoia, Luciano Zardi
• Journal: PloS one (PLoS One) Vol. 8 Issue 12 Pg. e82878 ( 2013) ISSN: 1932-6203 [Electronic] United States
• PMID: 24367567 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Antibodies
• Fibronectins
• Recombinant Proteins
• oncofetal fibronectin
• Uteroglobin

Topics

• Animals
• Antibodies (genetics, immunology, metabolism)
• Escherichia coli (genetics, metabolism)
• Fibronectins (immunology)
• Humans
• Immunohistochemistry
• Male
• Mass Spectrometry
• Mice
• Mice, SCID
• Protein Folding
• Recombinant Proteins (genetics, immunology, metabolism)
• Uteroglobin (chemistry, metabolism)