Coexpression of cytochrome P4502A6 and human NADPH-P450 oxidoreductase in the baculovirus system

Title

Coexpression of cytochrome P4502A6 and human NADPH-P450 oxidoreductase in the baculovirus system

Creator

Chen L P; Buters J T M; Hardwick J P; Tamura S; Penman B W; Gonzalez F J; Crespi C L

Publisher

Drug Metabolism and Disposition

Date

1997
1997-04

Description

Heterologous expression using baculovirus vectors has become a popular method for the production of catalytically active cytochrome P450s (CYPs). We have systematically optimized the multiplicity of infection (MOI) for a coinfection approach for the coexpression of CYP2A6 (viral vector designated v2A6) and NADPH-P450 oxidoreductase (OR; viral vector designated vOR) using Sf9 insect cells. A 3000-fold range of MOI was examined in stationary culture and stirred suspension culture. Surprisingly, our results Indicate that the best CYP2A6 catalytic activity (850-1300 pmol/ min/mg total lysate protein as measured by coumarin 7-hydroxylase activity) was obtained only when using a low MOI of v2A6 (1.5-3 x 10(-2)) and a vOR of 10- to 20-fold less, This activity was similar to 7- to 11-fold higher than the best activity obtained when infecting cells with v2A6 alone. At this level of coinfection, the P450 content ranged from 180 to 250 pmol/mg total lysate protein, and the NADPH cytochrome c reductase activity ranged from 350 to 520 nmol/min/mg total lysate protein. Increasing the MOI of both viruses to 50-fold higher resulted in lower overall activity with the optimum (250 pmol/min/mg total lysate protein) being seen earlier postinfection (60 vs. 72 hr). Increasing the MOI of vOR to levels comparable with those of v2A6, decreased coumarin 7-hydroxylase activity 14-fold. These results suggest that the best CYP2A6 catalytic activity depends on properly posttranslationally modified proteins accumulating in a right ratio as a result of primary, secondary, and possibly tertiary infection of both viruses. These results also suggest that high OR expression results in degradation of P450.

Subject

human; Pharmacology & Pharmacy; protein; enzymes; reductase; liver-microsomes; catalytic properties; cdna-directed expression; insect cells; nuclear polyhedrosis-virus; promoters; vector system

Identifier

n/a

Format

Journal Article or Conference Abstract Publication

URL Address

n/a

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Rights

Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).

Pages

399-405

Issue

4

Volume

25

Citation

Chen L P; Buters J T M; Hardwick J P; Tamura S; Penman B W; Gonzalez F J; Crespi C L, “Coexpression of cytochrome P4502A6 and human NADPH-P450 oxidoreductase in the baculovirus system,” NEOMED Bibliography Database, accessed October 24, 2021, https://neomed.omeka.net/items/show/8870.

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