Sonntag Michael H, Ibach Jenny, Nieto Lidia, Verveer Peter J, Brunsveld Luc
Laboratory of Chemical Biology, Department of Biomedical Engineering and Institute of Complex Molecular Systems, University of Technology, Den Dolech 2, 5612 AZ Eindhoven (NL).
Chemistry. 2014 May 12;20(20):6019-26. doi: 10.1002/chem.201304090. Epub 2014 Apr 3.
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.
迫切需要具有可选择性寻址标签的明确的人表皮生长因子（hEGF）构建体，以解决有关hEGF生物学的悬而未决的问题。开发了一种蛋白质工程方法，以获得多毫克量携带两个基于半胱氨酸的位点特异性正交标记位点的hEGF构建体。此外，还设计了一种位点选择性（去）保护和标记方法，该方法允许对这些hEGF构建体进行选择性修饰。具有三个天然二硫键的hEGF以半胱氨酸残基的形式表达额外的巯基，作为N和C末端的正交连接位点。以噻唑烷环的形式对N末端半胱氨酸单元进行临时保护，避免干扰蛋白质折叠，并能够与C末端的半胱氨酸残基一起进行顺序标记。基于由此产生的hEGF构建体，可以证明用各种分子探针进行顺序和位点特异性标记，从而产生具有特异性掺入荧光团或蛋白质货物以及天然细胞靶向和摄取特性的生物功能完全的hEGF。因此，这种新策略为高产获得hEGF以及对这种生长因子进行快速简便的位点特异性和多功能双标记提供了一条可靠途径。
