Escherichia coli expression and refolding of E/K-coil-tagged EGF generates fully bioactive EGF for diverse applications.
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 cell-based binding competition assays and by SPR-based biosensor experiments, indicated that the coil-tagged 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.
Le
Phuong U
PU
Biotechnology Research Institute, National Research Council of Canada, 6100 Royalmount Avenue, Montréal, Québec H4P2R2, Canada.
Lenferink
Anne E G
AE
Pinard
Maxime
M
Baardsnes
Jason
J
Massie
Bernard
B
O'Connor-McCourt
Maureen D
MD
D045744:Cell Line, Tumor; D002478:Cells, Cultured; D019281:Dimerization; D004815:Epidermal Growth Factor / Q000235:genetics* / Q000378:metabolism*; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D002479:Inclusion Bodies / Q000378:metabolism; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D020349:Surface Plasmon Resonance; D023822:Tissue Engineering; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism
C083605:K5 peptide; D010455:Peptides; D011994:Recombinant Proteins; D014443:Tyrosine; D004815:Epidermal Growth Factor; D066246:ErbB Receptors