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Escherichia coli expression and refolding of E/K-coil-tagged EGF generates fully bioactive EGF for diverse applications

NRC Institute for National Measurement Standards (INMS-IÉNM); National Research Council Canada; NRC Biotechnology Research Institute
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Protein Expression and Purification
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activity; analysis; bio; Biotechnology; Cell Culture; Cell Line,Tumor; Cell Proliferation; Cells; Cells,Cultured; chemistry; crossector; Dimerization; Epidermal Growth Factor; Escherichia coli; Gene Expression; genetics; Glioblastoma; Human; Humans; Inclusion Bodies; Ligands; metabolism; Nanoparticles; Peptides; pha; Phosphorylation; Protein; Protein Folding; Proteins; Receptor,Epidermal Growth Factor; Recombinant Proteins; scaffold; Surface Plasmon Resonance; Tags; Tissue Engineering; Tyrosine; EGF; E/K-coli; Refolding
Heterodimerizing peptides, such as the de novo designed E5/K5 peptide pair, have several applications including as tags for protein purification or immobilization. Recently, we demonstrated that E5-tagged epidermal growth factor (EGF), when bound to a K4 expressing adenovirus, promotes retargeting of the adenovirus to EGFR expressing target cells. In this study, we present the Escherichia coli expression, refolding and purification of human EGF fused with the E5-coil (E5-coil-EGF) or with the K5-coil (K5-coil-EGF). EGF receptor phosphorylation and cell proliferation assays demonstrated that the biological activity of the coil-tagged EGF versions was comparable to that of non-tagged EGF. Additionally, analysis of the binding of E5/K5-coil-EGF to cell surface EGFR or to soluble EGFR ectodomain, as measured by cellbased binding competition assays and by SPR-based biosensor experiments, indicated that the coiltagged EGF versions bound to EGFR with affinities similar to that of non-tagged EGF. Finally, we show that E-coil-tagged EGF, but not non-tagged EGF, can retarget a K-coil containing adenovirus to EGF receptor expressing glioblastoma tumor cells. Overall these results indicate that E. coli expression offers a practical platform for the reproducible production of fully biologically active E5/K5-coil-tagged EGF, and support applications of heterodimerizing coil-tagged ligands, e.g. the targeting of viruses or other entities such as nanoparticles to tumor cells, or growth factor immobilization on cell culture scaffolds for tissue engineering.
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