Figure 1. Protein engineering of split HRP (sHRP).

A. Schematic overview of the split HRP reporter. Two inactive fragments of HRP reconstitute into an active complex capable of producing a variety of enzymatic reaction products, visible by multiple modalities. The nonfluorescent Amplex Red is converted to fluorescent resorufin. Biotin-phenol is converted to a reactive radical that becomes covalently attached to neighboring proteins¹³. Tyramide signal amplification (TSA) substrates function analogously, except biotin is replaced with a fluorophore, such as Cy3. Diaminobenzidine (DAB) is converted to a colored and insoluble product that becomes electron-dense upon treatment with osmium tetroxide.

B. Overview of sHRP protein engineering. Structure-guided cut-site screening was followed by two rounds of yeast display directed evolution.

C. Overview of yeast display evolution of sHRPa (top) and sHRPb (bottom).

D. Fluorescence activated cell sorting (FACS) of yeast displaying sHRP fragments at various stages of evolution. Aga1p and 2p are cell surface mating proteins. Re-amplified pools of yeast were labeled and analyzed under matched conditions (1 min biotin-phenol labeling).

E. Crystal structure of HRP (PDB ID 1H5A). The sHRPa fragment is colored blue, and the sHRPb fragment is green.

F. Amplex UltraRed live cell labeling followed by immunostaining of HEK293T cells expressing sHRP fragments in the ER lumen.