Improving Split-HaloTag through Computational Protein Engineering

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Abstract 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 cpHaloΔ, 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 thermostability and activity of cpHaloΔ. These modifications decrease the time and substrate concentrations required for split-HaloTag-based assays and can expand their dynamic range and sensitivity.

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