Cysteinylated protein as reactive disulfide: an alternative route to affinity labeling.
Cysteinylated protein as reactive disulfide: an alternative route to affinity labeling.
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作为反应性二硫键的半胱氨酸化蛋白质：亲和标记的替代途径。
DOI：
10.1021/bc700330j
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发表时间：
2008
影响因子：
4.7
通讯作者：
Meares,ClaudeF
中科院分区：
文献类型：
--
作者：
Miao,Zheng;McCoy,MarkR;Singh,DimentD;Barrios,Brianda;Hsu,OliverL;Cheal,SarahM;Meares,ClaudeF
设计永久形成受体-配体复合物在化学、生物学和医学上有许多有前途的应用。抗体和其他蛋白质可以是合成配体如探针或药物的优良受体。因为蛋白质具有一系列亲核位点，所以在合成配体上放置亲电试剂以与大分子上的亲核试剂反应是标准做法。先前，我们已经使用半胱氨酸亲核试剂在抗体结合位点的外围的定点掺入，与弱亲电配体的化学设计配对，以产生特异性和永久性缀合的受体-配体对（Corneillie等人（2004）Bioconjugate Chem.15，1392-1402和其中的参考文献）。在果蝇S2细胞中蛋白质表达后，我们发现，正如经常观察到的那样，工程化的半胱氨酸被二硫键可逆地阻断为半胱氨酸单体（半胱氨酸化）。由于需要保留蛋白质中的其他二硫键，因此去除半胱氨酸单体需要一定的小心。在这里，我们报告，半胱氨酸化可以用来处理的半胱氨酸单体作为一个离去基团和蛋白质二硫化物作为亲电试剂具有特殊的亲和力硫醇的优势。合成两个带有巯基侧链的配体，并与半胱氨酸化的抗体Fab片段2D12.5 G54 C孵育，发现两个配体在生理条件下在几分钟内共价连接。即使在过量硫醇试剂存在下，连接也是稳健的。这种快速、特异性的缀合对于生物医学应用特别有意义。
Engineering the permanent formation of a receptor−ligand complex has a number of promising applications in chemistry, biology, and medicine. Antibodies and other proteins can be excellent receptors for synthetic ligands such as probes or drugs. Because proteins possess an array of nucleophilic sites, the placement of an electrophile on the synthetic ligand to react with a nucleophile on the macromolecule is a standard practice. Previously, we have used the site-directed incorporation of cysteine nucleophiles at the periphery of an antibody’s binding site, paired with the chemical design of weakly electrophilic ligands, to produce receptor–ligand pairs that conjugate specifically and permanently (Corneillie et al. (2004)Bioconjugate Chem. 15, 1392–1402 and references therein). After protein expression in Drosophila S2 cells, we found, as is frequently observed, that the engineered cysteine was reversibly blocked by disulfide linkage to a cysteine monomer (cysteinylated). Removal of the cysteine monomer requires some care because of the need to preserve other disulfide linkages in the protein. Here, we report that cysteinylation can be used to advantage by treating the cysteine monomer as a leaving group and the protein disulfide as an electrophile with special affinity for thiols. Two ligands bearing thiol side chains were synthesized and incubated with the cysteinylated antibody Fab fragment 2D12.5 G54C, with the finding that both ligands become covalently attached within a few minutes under physiological conditions. The attachment is robust even in the presence of excess thiol reagents. This rapid, specific conjugation is particularly interesting for biomedical applications.