Site-specific cross-linking of proteins through tyrosine hexahistidine tags.
The genetic addition of hexahistidine (H(6)) tags is widely used to isolate recombinant proteins by immobilized metal-affinity chromatography (IMAC). Addition of a tyrosine residue to H(6) 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(6)Y tags) were added to the amino terminus of the I28 immunoglobulin domain of the human cardiac titin. The H(6)Y-tagged I28 dimerized in the presence of excess Ni(2+) with a K(D) of 200 microM. Treatment of Ni(2+)-dimerized H(6)Y-I28 with an oxidant, monoperoxyphthalic acid (MMPP) or sodium sulfite, resulted in covalent protein multimerization through chelated Ni(2+)-catalyzed cross-linking of the Y residues engineered into the H(6) 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.
Stayner
R Scott
RS
Department of Bioengineering, 20 South 2030 East, Room 506, University of Utah, Salt Lake City, Utah 84112, USA.
Min
Dong-Joon
DJ
Kiser
Patrick F
PF
Stewart
Russell J
RJ
D001665:Binding Sites; D064211:Connectin; D003432:Cross-Linking Reagents / Q000737:chemistry*; D006639:Histidine; D006801:Humans; D015335:Molecular Probes / Q000138:chemical synthesis*; D009124:Muscle Proteins / Q000737:chemistry; D015202:Protein Engineering; D011494:Protein Kinases / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D014443:Tyrosine / Q000737:chemistry*
D064211:Connectin; D003432:Cross-Linking Reagents; D015335:Molecular Probes; D009124:Muscle Proteins; D011506:Proteins; C577285:TTN protein, human; D014443:Tyrosine; D006639:Histidine; D011494:Protein Kinases