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Titolo:
COEXPRESSION OF CYTOCHROME P4502A6 AND HUMAN NADPH-P450 OXIDOREDUCTASE IN THE BACULOVIRUS SYSTEM
Autore:
CHEN LP; BUTERS JTM; HARDWICK JP; TAMURA S; PENMAN BW; GONZALEZ FJ; CRESPI CL;
Indirizzi:
NIAID,IMMUNOL LAB,NIH,BLDG 10,ROOM 11N311 BETHESDA MD 20892 NCI,NIH BETHESDA MD 20892 NE OHIO UNIV,COLL MED ROOTSTOWN OH 44272
Titolo Testata:
Drug metabolism and disposition
fascicolo: 4, volume: 25, anno: 1997,
pagine: 399 - 405
SICI:
0090-9556(1997)25:4<399:COCPAH>2.0.ZU;2-T
Fonte:
ISI
Lingua:
ENG
Soggetto:
NUCLEAR POLYHEDROSIS-VIRUS; CDNA-DIRECTED EXPRESSION; HUMAN LIVER-MICROSOMES; INSECT CELLS; CATALYTIC PROPERTIES; VECTOR SYSTEM; REDUCTASE; PROMOTERS; ENZYMES; PROTEIN;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Citazioni:
37
Recensione:
Indirizzi per estratti:
Citazione:
L.P. Chen et al., "COEXPRESSION OF CYTOCHROME P4502A6 AND HUMAN NADPH-P450 OXIDOREDUCTASE IN THE BACULOVIRUS SYSTEM", Drug metabolism and disposition, 25(4), 1997, pp. 399-405

Abstract

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 (viralvector 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/mgtotal 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. 72hr). 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.

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Documento generato il 31/05/20 alle ore 13:33:21