Site-specific protection and dual labeling of human epidermal growth factor (hEGF) for targeting, imaging, and cargo delivery.
Well-defined human epidermal growth factor (hEGF) constructs featuring selectively addressable labels are urgently needed to address outstanding questions regarding hEGF biology. A protein-engineering approach was developed to provide access to hEGF constructs that carry two cysteine-based site-specific orthogonal labeling sites in multi-milligram quantities. Also, a site-selective (de)protection and labeling approach was devised, which allows selective modification of these hEGF constructs. The hEGF, featuring three native disulfide bonds, was expressed featuring additional sulfhydryl groups, in the form of cysteine residues, as orthogonal ligation sites at both the N and C termini. Temporary protection of the N-terminal cysteine unit, in the form of a thiazolidine ring, avoids interference with protein folding and enables sequential labeling in conjunction with the cysteine residue at the C terminus. Based on thus-generated hEGF constructs, sequential and site-specific labeling with a variety of molecular probes could be demonstrated, thus leading to a biological fully functional hEGF with specifically incorporated fluorophores or protein cargo and native cellular targeting and uptake profiles. Thus, this novel strategy provides a robust entry to high-yielding access of hEGF and rapid and easy site-specific and multifunctional dual labeling of this growth factor.
Laboratory of Chemical Biology, Department of Biomedical Engineering and Institute of Complex Molecular Systems, University of Technology, Den Dolech 2, 5612 AZ Eindhoven (NL).
Michael H
MH
Sonntag
Jenny
J
Ibach
Lidia
L
Nieto
Peter J
PJ
Verveer
Luc
L
Brunsveld
D003545:Cysteine / Q000737:chemistry* / Q000235:genetics; D003952:Diagnostic Imaging; D016503:Drug Delivery Systems; D004815:Epidermal Growth Factor / Q000008:administration & dosage* / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D008958:Models, Molecular; D015335:Molecular Probes / Q000737:chemistry; D015202:Protein Engineering; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000008:administration & dosage / Q000737:chemistry / Q000235:genetics; D053778:Thiazolidines / Q000737:chemistry*
D015335:Molecular Probes; D011993:Recombinant Fusion Proteins; D053778:Thiazolidines; D004815:Epidermal Growth Factor; D003545:Cysteine
drug delivery; growth factors; labeling; protein modifications; thiazolidines