Patent FR-2937976-A1 (FR)
Title: Culturing Saccharomyces cerevisiae yeast strain expressing human functional patched protein, comprises cloning sequences in yeast expression vector, transforming yeasts with vector and culturing yeasts on rich culture medium
Inventor: MUS VETEAU ISABELLE
IPC: C12P21/00
Filed: 2008-11-03
Abstract: Culturing a strain of Saccharomyces cerevisiaeyeast expressing the human functional patched protein, comprises (a) cloning of a first sequence encoding a polypeptide fused to the human patched protein e.g. one or more protease cleavage sites, and a second sequence having complementary DNA (cDNA) sequence encoding the human patched protein in the position 5' of the first sequence, in a yeast expression vector; (b) transforming the yeasts with the vector; and (c) culturing the yeasts on a rich culture medium. Culturing a strain of Saccharomyces cerevisiaeyeast expressing the human functional patched protein, comprises (a) cloning of a first sequence encoding a polypeptide fused to the human patched protein comprising (i) one or more protein fragments capable of binding on a separation support, (ii) a protein fragment capable of binding with at least one antibody, and (iii) one or more protease cleavage sites, and a second sequence consisting of complementary DNA (cDNA) sequence encoding the human patched protein in the position 5' of the first sequence, in a yeast expression vector; (b) transforming Saccharomyces cerevisiaeyeasts with the expression vector; and (c) culturing the obtained Saccharomyces cerevisiaeyeasts on a rich culture medium, where the culturing step is performed under conditions such that culture does not exceed a value of optical density of 8 at 600 nm and the medium is aerated with a ratio of volume of culture medium on the volume of air of 1:5 to 1:10 or with a flow of air introduced under pressure equal to 1-3 times the volume of the fermenter per minute. Independent claims are included for: (1) the Saccharomyces cerevisiaeyeast strain expressing the functional human patched protein obtained by the process; (2) a process for preparing membrane extracts of Saccharomyces cerevisiaeyeast comprising a functional patched protein receptor, comprising (a) mixing the yeasts expressing the protein receptor with a buffer comprising (i) a mixture of protease inhibitors comprising benzamidine (at least 2 mM), phenylmethylsulfonyl fluoride (PMSF) (at least 1 mM) and EDTA (at least 1 mM) and (ii) glass beads; (b) stirring the obtained mixture by vortex at a speed of 1500-2500 rpm for 10-20 minutes; (c) eliminating glass beads; (d) ultracentrifugation of the obtained supernatant (at least 18000 g) for 20 minutes; and (e) recovering the pellet constituted of membranes comprising the protein receptor; (3) an extracts of Saccharomyces cerevisiaemembranes comprising the functional human patched receptor obtained by process; (4) a process of screening the ligand of the human patched protein comprising: contacting the human patched receptor and molecules to be tested; recovering the human patched receptor optionally linked to molecules to be tested; detecting an interaction between the human patched receptor and the molecules to be tested; and selecting the molecules to be tested for which an interaction is measured. ACTIVITY : Cytostatic; Neuroprotective. MECHANISM OF ACTION : None given.