Abstract
Abstract Chemical labeling of proteins with synthetic molecular probes offers the possibility to probe the functions of proteins of interest in living cells. However, the methods for covalently labeling targeted proteins using complementary peptide tag-probe pairs are still limited, irrespective of the versatility of such pairs in biological research. Herein, we report the new CysHis tag-Ni(II) probe pair for the specific covalent labeling of proteins. A broad-range evaluation of the reactivity profiles of the probe and the CysHis peptide tag afforded a tag-probe pair with an optimized and high labeling selectivity and reactivity. In particular, the labeling specificity of this pair was notably improved compared to the previously reported one. This pair was successfully utilized for the fluorescence imaging of membrane proteins on the surfaces of living cells, demonstrating its potential utility in biological research.
Highlights
Methods for selectively labeling proteins with a synthetic molecular probe is an important research tool that facilitates the functional analysis of proteins in biological systems
Several peptide tag-based approaches have been devised for specific covalent labeling of proteins, and used in biological studies involving fluorescence imaging of proteins, cell functional analysis, and the design of antibody-drug conjugates.25 Compared with the enzyme-mediated protein labeling methods such as Halo tag and SNAP tag systems,[6] these chemical labeling methods benefit from the small molecular size of the tag-probe pair, which is unlikely to disturb protein functions, flexible probe design independent from the substrate specificity of enzymes, and high tolerability under various labeling conditions
We have recently demonstrated that histidine tag (His tag) peptide containing a cysteine residue (CysHis tag) underwent a rapid reaction with Ni(II) complexes (Figure 1)
Summary
Methods for selectively labeling proteins with a synthetic molecular probe is an important research tool that facilitates the functional analysis of proteins in biological systems. The oligo-histidine tag (His tag) is a representative epitope tag that has been widely used for the purification of recombinant proteins.[7] By exploiting its specific interaction with the Ni(II) complex, we and others have reported methods for the covalent labeling of His tag-fused proteins with a synthetic probe.[8,9] We have recently demonstrated that His tag peptide containing a cysteine residue (CysHis tag) underwent a rapid reaction with Ni(II) complexes (Figure 1). The overly reactive αchloroacetamide probe often induced non-specific labeling, hampering its wide use in fluorescence analysis of proteins Despite this problem, we have not fully optimized the reaction kinetics and labeling selectivity of this tag-probe pair, both of which are crucially important to achieve highly specific labeling of targeted tag-fused proteins in complicated biological contexts. The utility of this pair was demonstrated in the fluorescence imaging of membrane proteins on the surfaces of living cells
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