A yeast-based growth assay for the analysis of site-specific proteases. Export

@article{citeulike:2673622, abstract = {Many cellular and viral processes depend on site-specific proteolysis. Here, a genetic system for the identification of such proteases and inhibitors is described. The system utilizes the temperature- sensitive Saccharomyces cerevisiae CDC25-2 mutant strain and exploits the strict requirement of membrane localization of a constitutively active Ras mutant for the complementation of the yeast growth defect at the non-permissive temperature. Expression of a fusion protein in which a substrate peptide of the TEV protease separates a myristoylation signal from a constitutively active human Ras protein confers temperature insensitivity. Co-expression of the protease results in release of the Ras mutant from the membrane and growth arrest at the non-permissive temperature. This non-transcriptional assay represents a new approach to the in vivo analysis of site-specific proteases and may be a valuable alternative to existing methods. It has significant potential for the selection of inhibitors of cytoplasmic and membrane-associated proteases of biotechnical and clinical relevance.}, address = {Institut f\"{u}r Biologie III, Albert-Ludwigs-Universit\"{a}t, Sch\"{a}nzlestrasse 1, 79104 Freiburg, Germany. fabian.koehler@biologie.uni-freiburg.de}, author = {K\"{o}hler, F.}, citeulike-article-id = {2673622}, citeulike-linkout-0 = {http://view.ncbi.nlm.nih.gov/pubmed/12582261}, citeulike-linkout-1 = {http://www.hubmed.org/display.cgi?uids=12582261}, issn = {1362-4962}, journal = {Nucleic acids research}, keywords = {cleavage, ecoli, expression, protease, screening}, month = {February}, number = {4}, posted-at = {2008-04-15 15:42:35}, priority = {2}, title = {A yeast-based growth assay for the analysis of site-specific proteases.}, url = {http://view.ncbi.nlm.nih.gov/pubmed/12582261}, volume = {31}, year = {2003} }

TY - JOUR ID - citeulike:2673622 L3 - citeulike-article-id:2673622 N2 - Many cellular and viral processes depend on site-specific proteolysis. Here, a genetic system for the identification of such proteases and inhibitors is described. The system utilizes the temperature- sensitive Saccharomyces cerevisiae CDC25-2 mutant strain and exploits the strict requirement of membrane localization of a constitutively active Ras mutant for the complementation of the yeast growth defect at the non-permissive temperature. Expression of a fusion protein in which a substrate peptide of the TEV protease separates a myristoylation signal from a constitutively active human Ras protein confers temperature insensitivity. Co-expression of the protease results in release of the Ras mutant from the membrane and growth arrest at the non-permissive temperature. This non-transcriptional assay represents a new approach to the in vivo analysis of site-specific proteases and may be a valuable alternative to existing methods. It has significant potential for the selection of inhibitors of cytoplasmic and membrane-associated proteases of biotechnical and clinical relevance. IS - 4 JF - Nucleic acids research SN - 1362-4962 AD - Institut für Biologie III, Albert-Ludwigs-Universität, Schänzlestrasse 1, 79104 Freiburg, Germany. fabian.koehler@biologie.uni-freiburg.de TI - A yeast-based growth assay for the analysis of site-specific proteases. VL - 31 KW - cleavage KW - ecoli KW - expression KW - protease KW - screening AU - Köhler, F PY - 2003/02/15/ UR - http://view.ncbi.nlm.nih.gov/pubmed/12582261 ER -