A Highly Sensitive Assay for Proteases Using Staphylococcal Protein A Fused with Enhanced Green Fluorescent Protein

Abstract

  Enhanced green fluorescent protein (EGFP) was fused with staphylococcal protein A (SpA) and used as a substrate for proteases. An SpA-EGFP assay was done in three steps: (i) digestion of SpA-EGFP by proteases, (ii) addition of rabbit IgG immobilized on Sepharose beads, and (iii) measurement of the fluorescence intensity of supernatant. The assay was sensitive enough to measure picogram levels of trypsin and chymotrypsin, and may be applicable to various other proteases as one of the most sensitive methods.