Site-specific cross-linking of proteins through tyrosine hexahistidine tags

R. Scott Stayner, Dong Joon Min, Patrick F. Kiser, Russell J. Stewart*

*Corresponding author for this work

Research output: Contribution to journalArticle

23 Scopus citations

Abstract

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 H6 tags 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 (H 6Y 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 a KD of 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 H6 tag. 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 H 6 tag 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.

Original languageEnglish (US)
Pages (from-to)1617-1623
Number of pages7
JournalBioconjugate Chemistry
Volume16
Issue number6
DOIs
StatePublished - Nov 1 2005

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Biomedical Engineering
  • Pharmacology
  • Pharmaceutical Science
  • Organic Chemistry

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