The bioactivity and receptor affinity of recombinant tagged EGF designed for tissue engineering applications is defined by the nature and position of the tags.
For tissue engineering applications, growth factor immobilization on cell culture scaffolds bears the potential to stimulate cell proliferation while minimizing costs associated to soluble growth factor supply. In order to evaluate the potential of a de novo-designed heterodimerization peptide pair, namely the E and K coils, for epidermal growth factor (EGF) grafting on various scaffolds, production of coil-tagged EGF chimeras using a mammalian cell expression system as well as their purification have been performed. The influence of the type of coil (E or K) upon EGF bioactivity, assessed in an in vitro cell assay, was compared to that of the fragment crystallizable (Fc) domain of immunoglobulin G by monitoring phosphorylation of EGF receptor (EGFR) upon chimeric EGF exposure. Our results demonstrate that the type and the location of the tag have a strong impact on growth factor bioactivity (EC50 ranging from 5.5 to 63 nM). Additional surface plasmon resonance-based biosensor experiments were conducted to test the ability of captured chimeric EGF to bind to their receptor ectodomain in vitro. These experiments indicated that the oriented coiled-coil-mediated immobilization of EGF was significantly more efficient than a random approach as coil-tagged EGF displayed enhanced affinities for artificially dimerized EGFR ectodomain when compared to Fc-tagged EGF (apparent KD of 5 pM vs. 16 nM). Altogether, our results highly suggest that coil-tagged chimeras represent an attractive avenue for the oriented immobilization of growth factors for tissue engineering applications and that HEK293 cells offer a robust platform for their expression in a bioactive form.
Boucher
Cyril
C
Animal Cell Technology Group, Bioprocess Sector, Biotechnology Research Institute, National Research Council Canada, Montreal, Canada.
St-Laurent
Gilles
G
Loignon
Martin
M
Jolicoeur
Mario
M
De Crescenzo
Gregory
G
Durocher
Yves
Y
D001681:Biological Assay; D002460:Cell Line; D003001:Cloning, Molecular; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004815:Epidermal Growth Factor / Q000378:metabolism*; D066246:ErbB Receptors / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D010957:Plasmids / Q000235:genetics; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011961:Receptors, Fc / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism*; D020349:Surface Plasmon Resonance; D013816:Thermodynamics; D023822:Tissue Engineering
D011961:Receptors, Fc; D011993:Recombinant Fusion Proteins; D004815:Epidermal Growth Factor; D066246:ErbB Receptors