Improving split‐ HaloTag through computational protein engineering

Split‐HaloTag can be used to transform transient molecular interactions into permanent marks through chemical labeling, thereby enabling the recording of transient physiological events in individual cells. However, applications of split‐HaloTag‐based recorders can be limited by slow labeling rates. To address this issue, we have engineered an improved version of cpHalo∆, the larger fragment of the split‐HaloTag system. Using computational techniques, we identified stabilizing point mutations and designed a structured linker connecting the original N and C termini of the circular permutated protein, thereby significantly improving the thermostability and activity of cpHalo∆. These modifications decrease the time and substrate concentrations required for split‐HaloTag‐based assays and can expand their dynamic range and sensitivity.