| 摘要 |
This disclosure generally relates to components and methods of using a high throughput screening (HTS) systems for intracellular proteases, using Caspases as a prototype. Genetic systems are disclosed for monitoring exogenous caspase activation pathways in the yeast, Saccharomyces cerevisiae. The yeast-based cellular systems permit facile expression of proteases (e.g., caspase) and protease-activating proteins in combinations that reconstitute entire mammalian pathways in these simple eukaryotes. Among the assay methods integrated into the yeast system are cleavable reporter gene activators, in which protease-mediated cleavage activates a transcription factor. Exemplary systems rely, singly or in concert, on exogenous recombinant caspases and exogenous upstream activators of caspases to cleave a chimeric protein giving rise to a transcription factor which induces the expression of the LacZ and LEU2 genes. The activities of these genes result in colored cultures and impart the ability of the yeast to grow in leucine deficient media. The intensity of the color is measured by colorimetry and quantified with OD units. The OD units are directly proportional to the activity of the caspase in the system. The method of quantification is referred to as the "readout".
|
