Site-specific cross-linking of proteins through tyrosine hexahistidine tags.
Site-specific cross-linking of proteins through tyrosine hexahistidine tags.
复制标题
通过酪氨酸六组氨酸标签进行蛋白质的位点特异性交联。
DOI：
10.1021/bc050249b
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发表时间：
2005
期刊：
影响因子：
--
通讯作者：
Stewart,RussellJ
中科院分区：
文献类型：
--
作者：
Stayner,RScott;Min,Dong-Joon;Kiser,PatrickF;Stewart,RussellJ
The genetic addition of hexahistidine (H6) tags is widely used to isolate recombinant proteins by immobilized metal-affinity chromatography (IMAC). Addition of a tyrosine residue to H6tags enabled proteins to be covalently cross-linked under mild conditions in a manner similar to the natural, site-specific cross-linking of tyrosines into dityrosine. A series of seven hexahistidine tags with tyrosines placed in various positions (H6Y tags) were added to the amino terminus of the I28 immunoglobulin domain of the human cardiac titin. The H6Y-tagged I28 dimerized in the presence of excess Ni2+with aKDof 200 μM. Treatment of Ni2+-dimerized H6Y-I28 with an oxidant, monoperoxyphthalic acid (MMPP) or sodium sulfite, resulted in covalent protein multimerization through chelated Ni2+-catalyzed cross-linking of the Y residues engineered into the H6tag. The protein oligomerization was observed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE). The presence of dityrosine in the cross-linked proteins was confirmed by fluorescence emission at 410 nm. Proteins lacking the Y residue in the H6tag treated with the same oxidative conditions did not cross-link or exhibit dityrosine fluorescence, despite the presence of an endogenous Y residue. The method may have potential uses in other protein conjugation applications such as protein labeling and interfacial immobilization of proteins on artificial surfaces.
