Site-specific protection and dual labeling of human epidermal growth factor (hEGF) for targeting, imaging, and cargo delivery
| Autor: | Lidia Nieto, Jenny Ibach, MH Michael Sonntag, Peter J. Verveer, Luc Brunsveld |
|---|---|
| Přispěvatelé: | Chemical Biology |
| Jazyk: | angličtina |
| Rok vydání: | 2014 |
| Předmět: |
Diagnostic Imaging
Models Molecular Protein Folding Recombinant Fusion Proteins medicine.medical_treatment Cellular targeting Protein Engineering Catalysis Drug Delivery Systems growth factors medicine Humans Cysteine labeling Dual labeling Epidermal Growth Factor Chemistry Human epidermal growth factor Growth factor Organic Chemistry General Chemistry protein modifications Cell biology Biochemistry Molecular Probes Drug delivery drug delivery Protein folding thiazolidines Molecular probe |
| Zdroj: | Chemistry : A European Journal, 20(20), 6019-6026. Wiley-VCH Verlag |
| ISSN: | 0947-6539 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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