Maximizing the expression of mammalian cytochrome P-450 monooxygenase activities in yeast cells

Chimeras of the Human Cytochrome P450 1A Family Produced in Yeast

“…This is consistent with the observation that truncated mouse P450 1Al devoid of signal sequence is synthesised in a functional form in yeast although it is synthesized with a reduced efficiency (Cullin, 1992). In contrast, the deletion of the signal sequence of yeast CPR, which does not require the presence of membrane for folding, enhances sevenfold the expression level (Urban et al, 1990). Additionally, fusion of the signal sequence of yeast CPR to the E. coli P-glucuronidase renders the p-glucuronidase activity microsomal and decreases 50-fold its expression level (unpublished results).…”

Chimeras of the Human Cytochrome P450 1A Family Produced in Yeast

“…This is consistent with the observation that truncated mouse P450 1Al devoid of signal sequence is synthesised in a functional form in yeast although it is synthesized with a reduced efficiency (Cullin, 1992). In contrast, the deletion of the signal sequence of yeast CPR, which does not require the presence of membrane for folding, enhances sevenfold the expression level (Urban et al, 1990). Additionally, fusion of the signal sequence of yeast CPR to the E. coli P-glucuronidase renders the p-glucuronidase activity microsomal and decreases 50-fold its expression level (unpublished results).…”

Dimer Stabilization by SpyTag/SpyCatcher Coupling of the Reductase Domains of a Chimeric P450 BM3 Monooxygenase from Bacillus spp. Improves its Stability, Activity, and Purification

“…[29] Recent insights into structural modelling of the dimeric full-length holoenzyme reveal that the interface of the FAD regions comprise no cysteines forming possible disulfide bridges. [30,52] This fact agrees with previous observations where DTT did not disrupt the dimeric structure of the P450 BM3 holoenzyme. [12,40] Monomeric structure was only detected when the enzyme was incubated over a long time at high DTT concentrations (> 50 mM).…”

Human‐liver cytochromes P‐450 expressed in yeast as tools for reactive‐metabolite formation studies

“…Human P450 2C9 and 2C10 in yeast microsomes were found to be highly susceptible to inactivation by repeated freeze-thawing (25% loss upon each cycle) so that this procedure was avoided. In both transformed yeasts the respective levels of the endogenous microsomal electron-chain components, cytochrome b, (= 0.05 nmol/mg microsomal protein) and NADPH-P450 reductase (activity = 30 nmol cytochrome c reduced X mg microsomal protein-' X min-'), were in the same range as those previously reported for different S. cerevisiue wild strains (Yoshida et al, 1974;Urban et al, 1990;Stansfield et al, 1991). In addition, since the human P450 2C9 and 2C10 forms have been described to hydroxylate tolbutamide (Brian et al, 1989b;Veronesse et al, 1991), this activity was determined as a control activity for both enzymes.…”

Chimeras of the Human Cytochrome P450 1A Family Produced in Yeast

“…This is consistent with the observation that truncated mouse P450 1Al devoid of signal sequence is synthesised in a functional form in yeast although it is synthesized with a reduced efficiency (Cullin, 1992). In contrast, the deletion of the signal sequence of yeast CPR, which does not require the presence of membrane for folding, enhances sevenfold the expression level (Urban et al, 1990). Additionally, fusion of the signal sequence of yeast CPR to the E. coli P-glucuronidase renders the p-glucuronidase activity microsomal and decreases 50-fold its expression level (unpublished results).…”

Dimer Stabilization by SpyTag/SpyCatcher Coupling of the Reductase Domains of a Chimeric P450 BM3 Monooxygenase from Bacillus spp. Improves its Stability, Activity, and Purification

“…[29] Recent insights into structural modelling of the dimeric full-length holoenzyme reveal that the interface of the FAD regions comprise no cysteines forming possible disulfide bridges. [30,52] This fact agrees with previous observations where DTT did not disrupt the dimeric structure of the P450 BM3 holoenzyme. [12,40] Monomeric structure was only detected when the enzyme was incubated over a long time at high DTT concentrations (> 50 mM).…”

Human‐liver cytochromes P‐450 expressed in yeast as tools for reactive‐metabolite formation studies

“…Human P450 2C9 and 2C10 in yeast microsomes were found to be highly susceptible to inactivation by repeated freeze-thawing (25% loss upon each cycle) so that this procedure was avoided. In both transformed yeasts the respective levels of the endogenous microsomal electron-chain components, cytochrome b, (= 0.05 nmol/mg microsomal protein) and NADPH-P450 reductase (activity = 30 nmol cytochrome c reduced X mg microsomal protein-' X min-'), were in the same range as those previously reported for different S. cerevisiue wild strains (Yoshida et al, 1974;Urban et al, 1990;Stansfield et al, 1991). In addition, since the human P450 2C9 and 2C10 forms have been described to hydroxylate tolbutamide (Brian et al, 1989b;Veronesse et al, 1991), this activity was determined as a control activity for both enzymes.…”

Chimeras of the Human Cytochrome P450 1A Family Produced in Yeast

“…This is consistent with the observation that truncated mouse P450 1Al devoid of signal sequence is synthesised in a functional form in yeast although it is synthesized with a reduced efficiency (Cullin, 1992). In contrast, the deletion of the signal sequence of yeast CPR, which does not require the presence of membrane for folding, enhances sevenfold the expression level (Urban et al, 1990). Additionally, fusion of the signal sequence of yeast CPR to the E. coli P-glucuronidase renders the p-glucuronidase activity microsomal and decreases 50-fold its expression level (unpublished results).…”

Dimer Stabilization by SpyTag/SpyCatcher Coupling of the Reductase Domains of a Chimeric P450 BM3 Monooxygenase from Bacillus spp. Improves its Stability, Activity, and Purification

“…[29] Recent insights into structural modelling of the dimeric full-length holoenzyme reveal that the interface of the FAD regions comprise no cysteines forming possible disulfide bridges. [30,52] This fact agrees with previous observations where DTT did not disrupt the dimeric structure of the P450 BM3 holoenzyme. [12,40] Monomeric structure was only detected when the enzyme was incubated over a long time at high DTT concentrations (> 50 mM).…”

Human‐liver cytochromes P‐450 expressed in yeast as tools for reactive‐metabolite formation studies

“…Human P450 2C9 and 2C10 in yeast microsomes were found to be highly susceptible to inactivation by repeated freeze-thawing (25% loss upon each cycle) so that this procedure was avoided. In both transformed yeasts the respective levels of the endogenous microsomal electron-chain components, cytochrome b, (= 0.05 nmol/mg microsomal protein) and NADPH-P450 reductase (activity = 30 nmol cytochrome c reduced X mg microsomal protein-' X min-'), were in the same range as those previously reported for different S. cerevisiue wild strains (Yoshida et al, 1974;Urban et al, 1990;Stansfield et al, 1991). In addition, since the human P450 2C9 and 2C10 forms have been described to hydroxylate tolbutamide (Brian et al, 1989b;Veronesse et al, 1991), this activity was determined as a control activity for both enzymes.…”