Sequence-Specific 2-Cyanobenzothiazole Ligation

Abstract

The use of small, natural chemical reporters in conjunction with catalyst-free bioorthogonal reactions will greatly streamline protein labeling in a cellular environment with minimum perturbation to their function. Here we report the discovery of a 2-cyanobenzothiazole (CBT)-reactive peptide tag, CX10R7, from a cysteine-encoded peptide phage library using the phage-assisted interrogation of reactivity method. Fusion of CX10R7 with a protein of interest allows site-specific labeling of the protein with CBT both in vitro and on the surface of E. coli cells. Mutagenesis studies indicated that the reactivity and specificity of CX10R7 are attributed to the sequence environment, in which the residues surrounding cysteine help to stabilize the ligation product.

Scheme 1. Cysteine-CBT Ligation

Figure 1

Selective labeling of the CX10R7-tagged sfGFP by biotin-PEG₄-CBT. (a) Reaction scheme. The reaction was set up by incubating 5 μM of CX10R7-sfGFP (or sfGFP), 500 μM of biotin-PEG₄-CBT, and 1 mM of TCEP in PBS, pH 7.4, at 37 °C for 1 h. (b) Deconvoluted masses of the product mixtures. See Table S12 for mass calculations. (c) Streptavidin blot to probe selective biotinylation of CX10R7-sfGFP by biotin-PEG₄-CBT. Equal protein loading was verified by Coomassie blue staining.

Figure 2

Selective labeling of the CX10R7-tagged membrane protein OmpX on E. coli surface with biotin-PEG₄-CBT. (a) Labeling scheme. (b) Micrographs of E. coli expressing either OmpX-A7 or OmpX-CX10R7. The cells were treated with 500 μM biotin- PEG₄-CBT for 1 h before PBS wash and subsequent staining with streptavidin-Alexa Fluor 568 for 45 min. The last row shows the 3× digitally zoomed-in images. Scale bar = 2 μm.