Site-specific Dual-labeling Of Proteins Using Split Inteins

Protein modification is a versatile tool for investing the proteins· interaction, biological andstructure function. Nowadays, with the development of protein modification technology, multi-siteprotein modification has been used in many life science fields, especially in the studying ofprotein conformational distributions and dynamics. Some multi-site protein modification base onchemical and\or molecular biology methods have been developed, but there are still somedisadvantages respectively.The development of protein trans-splicing provides a new protein labeling method based onprotein trans-splicing which is mediated by split-intein. Split-intein is an intein which splits inspecific internal site and two parts of the intein (N-intein and C-intein) encoded by two separateopen reading frames located apart in the genome, respectively. In the process of splicing, the N-intein and C-intein rapidly self-associate and reconstitute protein trans-splicing activity, exteinswith a peptide bond to produce the mature host protein, and the N-intein and C-inteinautocatalytically self-excise.Novel split-inteins SG-S1, SX-S1, TX-S11and TE3-S11were selected for the studies in thiswork. The N-intein of the S1split-inteins is only11-aa in length, and the C-intein of the S11split-inteins is only6-aa in length, thus they can easily be chemically produced to be added fluorescentlabels to the N-terminus or C-terminus of recombinant proteins. Intein-catalyzed protein trans-splicing reaction was mediated by two different split-inteins (S1and S11) between the targetprotein diUb and other two proteins carrying the desired labeling group respectively.The reactionproducts were detected by fluorescence scanning, SDS-PAGE and mass spectrometry finally.The results showed that diUb (ubiquitin dimer) protein was site-specifically labeled at the N-and C-terminal with two different fluoresceins successfully. The method in this paper has manyadvantages,such as specifity, generality, easily operation and so on. It is no longer confined to traditional amino acid-specific labeling. The potential application of split-inteins for multi-sitespecific protein labeling has led to the development of many applications in the fields of proteinengineering and research of protein dynamics.