Experience

Publications

• Biotechnology, Biosensors, and Beer: The measurement of proteases relevant to brewing

  Proceedings of the American Society of Brewing Chemists

  Proteases are responsible for the cleavage and breakdown of other proteins. Two proteases relevant to the brewing industry are the yeast proteinase A (PrA) and the Aspergillus niger prolylendoprotease (ANPEP). Using technology developed in our lab, we have engineered substrate-specific, fluorescent biosensors that detect these proteases using only a drop of beer. Originating from yeast, PrA is secreted into beer during stressful fermentation conditions like high gravity or poor nutrition. PrA degrades foam-positive proteins and decreases head retention in the final product. With our PrA biosensor, we have demonstrated that PrA activity is affected by fermentation temperature in a strain-dependent manner. Ultimately, yeast strains that show little to no PrA activity may be ideal for bottle or cask conditioning. ANPEP is a purified protease added by the brewer during fermentation to reduce chill haze and gluten content. Several biosensors were designed to measure ANPEP activity against specific protein sequences found in hordein or LTP-1. By designing different biosensors with physiologically relevant targets, we hope to provide a tool to measure ANPEP activity and to gain insight into substrates that may affect beer production.

• Biotechnology, Biosensors, and Beer: The measurement of proteases relevant to brewing

  Proceedings of the American Society of Brewing Chemists

  Proteases are responsible for the cleavage and breakdown of other proteins. Two proteases relevant to the brewing industry are the yeast proteinase A (PrA) and the Aspergillus niger prolylendoprotease (ANPEP). Using technology developed in our lab, we have engineered substrate-specific, fluorescent biosensors that detect these proteases using only a drop of beer. Originating from yeast, PrA is secreted into beer during stressful fermentation conditions like high gravity or poor nutrition. PrA degrades foam-positive proteins and decreases head retention in the final product. With our PrA biosensor, we have demonstrated that PrA activity is affected by fermentation temperature in a strain-dependent manner. Ultimately, yeast strains that show little to no PrA activity may be ideal for bottle or cask conditioning. ANPEP is a purified protease added by the brewer during fermentation to reduce chill haze and gluten content. Several biosensors were designed to measure ANPEP activity against specific protein sequences found in hordein or LTP-1. By designing different biosensors with physiologically relevant targets, we hope to provide a tool to measure ANPEP activity and to gain insight into substrates that may affect beer production.

• Shroom2 regulates contractility to control endothelial morphogenesis.

  Molecular Biology of the Cell

  The intrinsic contractile, migratory, and adhesive properties of endothelial cells are central determinants in the formation of vascular networks seen in vertebrate organisms. Because Shroom2 (Shrm2) is expressed within the endothelium, is localized to cortical actin and cell-cell adhesions, and contains a conserved Rho kinase (Rock) binding domain, we hypothesized that Shrm2 may participate in the regulation of endothelial cell behavior during vascular morphogenesis. Consistent with this hypothesis, depletion of Shrm2 results in elevated branching and sprouting angiogenic behavior of endothelial cells. This is recapitulated in human umbilical vein endothelial cells and in a vasculogenesis assay in which differentiated embryonic stem cells depleted for Shrm2 form a more highly branched endothelial network. Further analyses indicate that the altered behavior observed following Shrm2 depletion is due to aberrant cell contractility, as evidenced by decreased stress fiber organization and collagen contraction with an increase in cellular migration. Because Shrm2 directly interacts with Rock, and Shrm2 knockdown results in the loss of Rock and activated myosin II from sites of cell-cell adhesion, we conclude that Shrm2 facilitates the formation of a contractile network within endothelial cells, the loss of which leads to an increase in endothelial sprouting, migration, and angiogenesis.

• Biotechnology, Biosensors, and Beer: The measurement of proteases relevant to brewing

  Proceedings of the American Society of Brewing Chemists

  Proteases are responsible for the cleavage and breakdown of other proteins. Two proteases relevant to the brewing industry are the yeast proteinase A (PrA) and the Aspergillus niger prolylendoprotease (ANPEP). Using technology developed in our lab, we have engineered substrate-specific, fluorescent biosensors that detect these proteases using only a drop of beer. Originating from yeast, PrA is secreted into beer during stressful fermentation conditions like high gravity or poor nutrition. PrA degrades foam-positive proteins and decreases head retention in the final product. With our PrA biosensor, we have demonstrated that PrA activity is affected by fermentation temperature in a strain-dependent manner. Ultimately, yeast strains that show little to no PrA activity may be ideal for bottle or cask conditioning. ANPEP is a purified protease added by the brewer during fermentation to reduce chill haze and gluten content. Several biosensors were designed to measure ANPEP activity against specific protein sequences found in hordein or LTP-1. By designing different biosensors with physiologically relevant targets, we hope to provide a tool to measure ANPEP activity and to gain insight into substrates that may affect beer production.

• Shroom2 regulates contractility to control endothelial morphogenesis.

  Molecular Biology of the Cell

  The intrinsic contractile, migratory, and adhesive properties of endothelial cells are central determinants in the formation of vascular networks seen in vertebrate organisms. Because Shroom2 (Shrm2) is expressed within the endothelium, is localized to cortical actin and cell-cell adhesions, and contains a conserved Rho kinase (Rock) binding domain, we hypothesized that Shrm2 may participate in the regulation of endothelial cell behavior during vascular morphogenesis. Consistent with this hypothesis, depletion of Shrm2 results in elevated branching and sprouting angiogenic behavior of endothelial cells. This is recapitulated in human umbilical vein endothelial cells and in a vasculogenesis assay in which differentiated embryonic stem cells depleted for Shrm2 form a more highly branched endothelial network. Further analyses indicate that the altered behavior observed following Shrm2 depletion is due to aberrant cell contractility, as evidenced by decreased stress fiber organization and collagen contraction with an increase in cellular migration. Because Shrm2 directly interacts with Rock, and Shrm2 knockdown results in the loss of Rock and activated myosin II from sites of cell-cell adhesion, we conclude that Shrm2 facilitates the formation of a contractile network within endothelial cells, the loss of which leads to an increase in endothelial sprouting, migration, and angiogenesis.

• Engineering a biosensor to detect a secreted yeast protease in beer

  Proceedings of the Master Brewers Association of the Americas

  Proteases are responsible for the cleavage and breakdown of other proteins. Previous studies have demonstrated that the yeast protease, proteinase A (PrA), also called saccharopepsin, is secreted in beer during fermentation. This protease hypothetically degrades foam-promoting proteins, thus decreasing head retention of the beer. Because the concentration of secreted PrA during fermentation has been correlated with yeast viability and proper nutrition, the precise measurement of PrA activity is important for quality control. Previous assays to measure PrA activity have demonstrated non-specificity, low sensitivity, and high cost. Therefore, we have developed a novel, genetically encoded PrA sensor using engineered antibodies that fluoresce after cleavage by PrA. Using less than a drop of beer as the source of PrA, we can measure PrA activity over time. Thus far, we have demonstrated that mechanical disruption of yeast and prolonged storage of yeast increases the activity of PrA. In the future, we hope to use our PrA sensor to measure PrA activity induced by variables such as different yeast strains, improper handling of yeast, and prolonged back slopping. In addition, our platform will allow us to develop sensors for additional yeast proteases that might be relevant to the brewing industry.