Site specific labeling of two proteins in one system by atypical split inteins

Atypical inteins have been used for protein site-specific labeling and modification. S1 or S11 intein contain a small N-intein or C-intein which can be chemically synthesized and added with desired labels or modifications. However, seldom reports have been found to develop multi-protein specific labeling in one system at the same time. Here, we established a specific labeling method for two proteins based on three pairs of atypical inteins, RBS1 (Rma DnaB S1)/SGS1 (Ssp GyrB S1) intein, TE3S11 (Ter DnaE-3 S11)/SGS11 (Ssp GyrB S11) intein, and RBS1/TE3S11 intein. Firstly, intein fragments were respectively fused with model proteins, expressed and purified as precursors. The non-cross reactivity between inteins was then determined by splicing reactions analysis through Western-blot. Finally, the model proteins were specifically labeled with fluorescent groups in one system mediating these intein pairs, and can be labeled even in the cell lysate. Our results for the first time report a method labeling the N/C-terminal of two proteins altogether in the same system based on above intein pairs with a marked splicing efficiency, which could potentially be used for protein structural and functional research or some specific applications.