A highly sensitive assay for proteases using staphylococcal protein A fused with enhanced green fluorescent protein.
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.
Fujino
Hiroyoshi
H
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido, Ishikari-Tobetsu, Japan.
Aoki
Takashi
T
Watabe
Hiroyuki
H
D015415:Biomarkers; D002918:Chymotrypsin / Q000032:analysis; D010450:Endopeptidases / Q000032:analysis*; D005453:Fluorescence; D049452:Green Fluorescent Proteins; D007074:Immunoglobulin G / Q000737:chemistry; D008164:Luminescent Proteins / Q000138:chemical synthesis*; D011993:Recombinant Fusion Proteins; D012685:Sepharose; D013205:Staphylococcal Protein A; D014357:Trypsin / Q000032:analysis
D015415:Biomarkers; D007074:Immunoglobulin G; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; D049452:Green Fluorescent Proteins; D012685:Sepharose; D010450:Endopeptidases; D002918:Chymotrypsin; D014357:Trypsin