| Autor: |
Mitchell AC; Department of Bioengineering, Stanford University , Stanford, California 94305, United States., Alford SC; Department of Bioengineering, Stanford University , Stanford, California 94305, United States., Hunter SA; Cancer Biology Program, Stanford University , Stanford, California 94305, United States., Kannan D; Department of Bioengineering, Stanford University , Stanford, California 94305, United States., Parra Sperberg RA; Department of Bioengineering, Stanford University , Stanford, California 94305, United States., Chang CH; Department of Bioengineering, Stanford University , Stanford, California 94305, United States., Cochran JR; Department of Bioengineering, Stanford University , Stanford, California 94305, United States.; Cancer Biology Program, Stanford University , Stanford, California 94305, United States.; Department of Chemical Engineering, Stanford University , Stanford, California 94305, United States. |
| Abstrakt: |
Dysregulated activity of the protease matriptase is a key contributor to aggressive tumor growth, cancer metastasis, and osteoarthritis. Methods for the detection and quantification of matriptase activity and inhibition would be useful tools. To address this need, we developed a matriptase-sensitive protein biosensor based on a dimerization-dependent red fluorescent protein (ddRFP) reporter system. In this platform, two adjoining protein domains, connected by a protease-labile linker, produce fluorescence when assembled and are nonfluorescent when the linker is cleaved by matriptase. A panel of ddRFP-based matriptase biosensor designs was created that contained different linker lengths between the protein domains. These constructs were characterized for linker-specific cleavage, matriptase activity, and matriptase selectivity; a biosensor containing a RSKLRVGGH linker (termed B4) was expressed at high yields and displayed both high catalytic efficiency and matriptase specificity. This biosensor detects matriptase inhibition by soluble and yeast cell surface expressed inhibitor domains with up to a 5-fold dynamic range and also detects matriptase activity expressed by human cancer cell lines. In addition to matriptase, we highlight a strategy that can be used to create effective biosensors for quantifying activity and inhibition of other proteases of interest. |
