Use of Recombinant Peptide as Substrate in FRET Based Protease Assays.

Proteases are enzymes that catalyze the breakdown of peptide bonds. They are essential to all physiologic and pathological processes. Some protease deficiencies are closely associated with certain disease conditions, such as alpha-1-Antitrypsin deficiency in COPD, Antithrombin III deficiency in clotting disorders, and ADAMTS13 deficiency in thrombotic thrombocytopenic purpura (TTP). Measurement of protease activity is of great interest in research, drug discovery and development, and for use as diagnostic or prognostic markers in disease states.

Traditional protease assay methods involve multiple steps and are very time-consuming. Recently, fluorescence energy transfer (FRET) based protease assays have become popular. These assays eliminate gel electrophoresis, radioactivity, and the need for special skills or complicated instruments. They require only one step incubation and allow high throughput screening of a large number of samples.

Generation of pure, reliable, high quality substrate is essential to the development and improvement of such assays. Most FRET based assays use synthesized peptide substrates, the size of which is restricted by the limitations of chemical synthesis. These substrates are usually labeled with a pair of two different fluor-quencher molecules. However, when two identical fluorescent molecules are in close proximity, their fluorescence emission can also be quenched. This phenomenon of autoquenching has been widely used in protein structural studies and even in single-molecule structural studies, but has never been used to produce FRET substrates. We hypothesized that this mechanism can be applied to the production of recombinant FRET substrates. We used the ADAMTS13 - von Willebrand factor (VWF) system to test this hypothesis. ADAMTS13 is a metalloproteinase that cleaves VWF between Y1605 and M1606. Kokame et al. have shown that the smallest effective substrate is a 73 amino acid (AA) peptide from the VWF A2 domain. A recombinant 73 AA peptide was generated using Qiagen pQE-100 His Tag vector and M15 E. coli expression system. Two native amino acids, Q1599 and P1611, which flank the ADAMTS13 cleavage site, were mutated to cysteines. These two cysteines were subsequently labeled with fluorescein molecules. The separation of these two fluoresceins by ADAMTS13 cleavage generated measurable fluorescence increase. There was a steady increase of fluorescence over time during the incubation of this recombinant peptide with pooled normal plasma (PNP) used as the source of ADAMTS13. There was only minimal change in fluorescence during incubation with heat inactivated PNP and plasma samples from known TTP patients with absent ADAMTS13 activity. The enzyme kinetic assay yielded an excellent linear relationship between the rates of fluorescence increase and ADAMTS13 enzyme concentration. Our study has provided proof of principle that fluorogenic recombinant peptides can be generated for FRET based protease assays.