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.
Highlights
Fibronectins (FNs) are high molecular mass adhesive glycoproteins of the extracellular matrix (ECM)
L19-UG cDNA Construct and Protein Expression The cDNA sequence encoding the scFv L19 protein was provided by Shine Gene Molecular Biotech (Shanghai, China) and the cDNA sequence encoding the human fusion protein L19-UG, which was optimized for expression in E. coli and cloned into the pUC57 vector, was provided by GenScript (Piscataway, NJ)
We found that the best conditions for L19-UG expression were as follows: 0.2 mM IPTG, induction temperature of 18uC, incubation time of 72 hours and supplementation with 1% (v/v) Triton-X100
Summary
Fibronectins (FNs) are high molecular mass adhesive glycoproteins of the extracellular matrix (ECM). FNs are widely distributed in normal tissues and body fluids and are involved in several processes such as cell adhesion and migration, maintenance of normal cell morphology, cell growth and differentiation [1,2]. Fibronectins are encoded by a single gene localized on chromosome 2 [3], but different isoforms arise from the alternative splicing of the pre-mRNA, a process that for some ECM proteins, including fibronectin, is modulated by cytokines and extracellular/ intracellular pH [4,5,6]. The demonstration that murine monoclonal antibodies to B-FN injected into tumorbearing mice selectively accumulate in neoplastic lesions [13] prompted the generation of high-affinity human recombinant antibodies for therapeutic and diagnostic purposes [14,15,16,17,18,19,20,21]
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