Expression Constructs CYP75A31 was cut from the TOPO vector using Bam HIand EcoRI, then ligated into the pYeDP60 vector selleck chemicals Imatinib for expression in yeast. Yeast Expression and microsome preparation The yeast strain Saccharomyces cerevisiae WAT11, engi neered Inhibitors,Modulators,Libraries to over express the P450 reductase isoform ATR1 from Arabidopsis thaliana when induced with galactose, was used for the expression. Transforma tion with the pYeDP60 expression construct was per formed as previously described by Gietz et al. Propagation of yeast cells and preparation of micro somes was done as described by Pompon et al. with some modifications. Liquid SGlu, 50 ml, was inoculated by a single colony from a SGlu plate and grown at 30 C for 48 h. The culture was then transferred to 200 ml YPGlu medium, containing 20 g l glucose, and grown at 30 C for 24 h.
The yeast cells were spun down and re suspended in YPGal medium containing 20 g l galactose for induction of microsomes at 16 C for 24 h. Microsomes were isolated Inhibitors,Modulators,Libraries in the fol lowing way The yeast culture was centrifuged and the pellet re suspended in 50 ml TEK, centrifuged at 6 100 g for 3 min and the pellet re sus pended in 2 ml extraction buffer. Glass beads were added, and the suspension was shaken in an automatic shaker 4 2 min at a vibration frequency of 30. Between two shaking cycles the suspension was placed on ice for 3 min. Portions of 10 ml extraction buffer was added to the beads 4 times, shaken and decanted to retrieve the microsomes. Extraction buffer was centrifuged for 15 min at 6 100 g, the supernatant was filtered, and MgCl2 added to a final concentration of 50 mM in order to precipitate the microsomes.
The suspen Inhibitors,Modulators,Libraries sion was placed on ice for approximately 1 h before cen trifugation at 12 500 g for 20 min. The pellet was dissolved in 1. 0 to 1. 5 ml TEG and homogenized using a Teflon pestle. Work was carried out on ice, all buffers solutions and centrifuge were pre cooled to 4 C. CYP75A31 Enzyme assays Several compounds were tested as potential substrates for CYP75A31. Microsomes isolated from yeast CYP75A31 transformants were incubated in 0. 1 M sodium phosphate buffer, pH 7. 0 containing 1. 0 mM NADPH, or without NADPH. The assay mixture was equilibrated Inhibitors,Modulators,Libraries for 2 min at 27 C prior to starting the reaction by addition of microsomes. Concentration Inhibitors,Modulators,Libraries of substrate in the assays ranged between 20 to 100 uM. Total volume of assay was 200 ul.
After 10 to 30 min the reaction was stopped by adding 75 ul of acetonitrile concentrated HCl. Precipitated pro teins were removed by a 10 min centrifugation. selleck chemical Bicalutamide the supernatant was used directly for HPLC and MS analysis to assess product formation and substrate con sumption. To validate that hydroxylations occurred due to CYP75A31 activity, assays were run with a micro some preparation made from WAT11 transformed with the pYeDP60 vector without any insertions.