Functional High Level Expression of Cytochrome P450 CYP2D6 Using Baculoviral Expression Systems

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS

Vol. 328, No. 1, April 1, pp. 143–150, 1996Article No. 0154

Functional High Level Expression of Cytochrome P450CYP2D6 Using Baculoviral Expression Systems

Mark J. I. Paine,* David Gilham,* Gordon C. K. Roberts,† and C. Roland Wolf*,1

*Biomedical Research Centre, Ninewells Hospital and Medical School, University of Dundee, Dundee, DD1 9SY, Scotland;and †Biological NMR Centre, Medical Sciences Building, University of Leicester, Leicester, LE1 9HN, United Kingdom

Received October 20, 1995, and in revised form January 18, 1996

Key Words: CYP2D6; NADPH-cytochrome P450 re-ductase; baculovirus expression; coexpression; drugCytochrome P-450 CYP2D6 plays a central role inmetabolism.the metabolism of many widely used therapeutic

drugs including b-adrenergic antagonists, antiar-rhythmics, and tricyclic antidepressants. Recombi-nant baculoviruses have been constructed con-taining the full-length human CYP2D6 cDNA and Cytochrome P450’s belong to a superfamily of en-used to express CYP2D6 in Spodoptera frugiperda

zymes which catalyze the hydroxylation of a broad(Sf9) cells. High levels of recombinant protein havespectrum of xenobiotic chemicals and procarcinogensbeen produced using either polyhedrin or basic pro-(1, 2). Cytochrome P450 CYP2D6 plays a crucial roletein promoters (0.05–0.20 nmol/mg cell protein; 0.05–in the metabolism of several widely used therapeutic0.15 nmol/liter). The enzyme is catalytically active to-agents including cardiovascular drugs, b-adrenergicward CYP2D6 substrates such as bufuralol and meto-blocking agents, tricyclic antidepressents, and miscel-prolol. In order to optimize catalytic activity humanlaneous compounds such as methoxy amphetamine, co-reductase was coexpressed with CYP2D6 in Sf9 cells;deine, and dextromethorphan (3). In addition, CYP2D6reductase activity was in the region of 1000–1500is highly polymorphic, particularly among Caucasiansunits per mg cell protein, while spectrally activewhere 5–10% of the populace are deficient in this en-CY2D6 was in the range 10–20 pmol/mg cell protein.

The Km and Kcat values for bufuralol metabolism were zyme. This can lead to adverse drug reactions in indi-estimated as 4.7 mM and 12.23 min01, respectively. The viduals who are poor metabolizers (4). Genetic polymor-use of the conventional very late promoters such as phism of CYP2D6 has been linked to susceptibility tothe polyhedrin promoter generate a large proportion cancer and an absence of CYP2D6 enzyme has beenof inactive CYP2D6. The problem was to a degree cir- associated with increased susceptibility to Parkinson’scumvented using the ‘‘late’’ basic promoter which is disease (7, 23, 24).active earlier in the baculovirus infection cycle. The In view of the importance of CYP2D6 in metabolizingyield of functional CYP2D6 was at least as high as a wide range of therapeutic agents, it is important towith very late promoters, but the proportion of inac- define in more detail how the structure of the enzymetive protein was reduced. Bufuralol hydroxylase ac- relates to its substrate specificity. Most CYP2D6 sub-tivity could be measured directly by HPLC analysis strates contain a basic nitrogen atom, which is as-of cell culture media supplemented with bufuralol, sumed to be protonated and to interact with a nega-and we have developed a plate assay system which

tively charged atom in the substrate binding site (8–provides a simple method for the analysis of drug10). On the basis of computer modelling programs, themetabolism reactions using Sf9 cells. Expression us-distance between the basic nitrogen atom and the siteing baculovirus provides a valuable source of func-of attack is thought to be somewhere between 5 and 7 Ational CYP2D6 for work aimed at elucidating the(9, 10). However, detailed information on the molecularstructure and function of the enzyme. q 1996 Academicinteractions of CYP2D6 and its substrates is largelyPress, Inc.unknown due to the absence of X-ray structures forCYP2D6 or any other membrane bound cytochromeP450. To date, only the crystal structures of the soluble1 To whom correspondence and reprint requests should be ad-

dressed. Fax: (01382) 669993. bacterial cytochromes P450 cam (11), P450 terp (26),

1430003-9861/96 $18.00Copyright q 1996 by Academic Press, Inc.All rights of reproduction in any form reserved.

/ 6b15$$9360 02-27-96 10:11:22 arca AP: Archives

144 PAINE ET AL.

digested out of pMP201 with EcoRI and ligated into a unique EcoRIP450 eryF (42), and the P450 domain of BM3 (12) havesite adjacent to the promoter PH in the baculoviral transfer vectorbeen solved.pVL1392 (Pharmingen) to create pMP522. The same CYP2D6 DNA

Heterologous protein expression can facilitate the fragment was also ligated into the EcoRI site in pAcMP2 adjacentanalysis of protein function; however, there are inher- to the core protein promoter (Pcor), to create pMP523. Plasmid p2Bac

(Pharmacia) was used to coexpress CYP2D6 and human NADPHent difficulties associated with the high level expres-cytochrome-P450 reductase. The cDNA for human reductase was assion of hydrophobic membrane proteins such as cyto-described previously (14). The full-length cDNA was cloned into thechrome P450’s. Although Escherichia coli has beenunique BamHI/BglII sites adjacent to the PH promoter in P2Bac to

used successfully to produce some P450’s, including create the construct pMP524. CYP2D6 cDNA was ligated into StuI/CYP2D6 (27), modification of the cDNA is required XbaI sites downstream of the P10 promoter in pMP524 to create the

dual expression transfer vector pMP525. Transfer vectors pMP522,(13). The baculovirus/Sf92 system has been used to ex-pMP523, pMP524, and pMP525 were used to produce, respectively,press several P450’s including CYP2A1 (39), CYP3A4the recombinant baculoviruses b2D6PH, b2D6cor , bRED, and bR2D6(33), and CYP2E1 (38) at levels up to 3% of total pro- through recombination with linearized AcRP23.lacZ DNA (Phar-

tein. The system has also been used for high level ex- mingen). For this, transfer vectors were cotransfected along withpression of P450 reductase (29). More recently, using AcRP23.lacZ DNA into Sf9 cells using a BaculoGold expression sys-

tem (Pharmingen) according to the manufacturer’s instructions.a single transfer vector, CYP3A4 has been coexpressedSf9 cell culture and recombinant protein expression. Sf9 cellswith reductase in Sf9 cells to produce a recombinant

were maintained at 277C in TNM-FH media (15). For expression,P450 enzyme system with catalytic activity comparableSf9 cells were infected with virus at a multiplicity of infection ofto that found in human liver microsomes (28). We have between 1 and 10 plaque forming units per cell. Hemin chloride (2.5

therefore examined the use of baculovirus as a means mg/ml) or d-amino levulinic acid (0.5 mM) was added to the cultureof producing functional CYP2D6 for structure/function media to compensate for low endogenous levels of haem in insect

cells. Cells were harvested 48–72 h after infection, washed withanalysis and investigate the possibility of optimizingPBS, and resuspended in 50 mM potassium phosphate, 20% glycerol,functional activity by increasing cellular P450 reduc-1 mM DTT, 1 mM EDTA, pH 7.7. All manipulations were carried outtase content. In this paper we describe the high level at 47C. Crude cell lysates were produced by brief sonication, and

expression of CYP2D6 in baculovirus and its coexpres- the P450 content was determined by measurement of CO-reducedspectra (16). CYP2D6 was solubilized by adding Emulgen 911 to 1%sion with human NADPH-cytochrome P450 reductasev/v, cells were homogenized with 10–20 strokes in a tightly fittingto produce a functional system capable of efficientlyglass Dounce homogenizer, and cell debris was pelleted at 38,000gmetabolizing 2D6 substrates.for 30 min. Protein concentrations were measured using the Bradfordmethod (22) against bovine serum albumin standards. Sodium dode-cylsulphate–polyacrylamide gel electrophoresis (SDS–PAGE) andMATERIALS AND METHODSWestern blotting was carried out using 10% gels (41).

Chemicals and reagents. Chemicals were from Sigma. The sub- Enzyme assays. The bufuralol 1*-hydroxylation activities ofstrates ({)-bufuralol, ({)-metoprolol and standards 1*-hydroxy bu- CYP2D6 were determined according to the general method of Kron-furalol, O-desmethyl metoprolol, and a-hydroxy-metoprolol were bach et al. (18) with modifications. For the assay reactions, 0.05–0.5kindly provided by Dr. Steve Clarke (Smith Kline Beecham, UK). mg of Sf9 cell extracts were incubated in 300-ml reactions containingEnzymes used in cloning were from Boehringer Mannheim or New 50 mM potassium phosphate (pH 7.4), 10 mM ({)-bufuralol, and anEngland Biolabs. Rat liver NADPH P450 reductase used in these NADPH generating system (2.5 mM glucose-6-phosphate, 0.5 mMexperiments was purified as described previously (14). Yeast micro- NADP/, 2 units/ml glucose-6-phosphate dehydrogenase). Where indi-somes expressing recombinant CYP2D6 were kindly provided by cated, rat NADPH reductase was included to compensate for lowMonica Bandera (Biomedical Research Center, Dundee). endogenous insect cell reductase activity. Sf9 cell extracts were pre-

Baculovirus constructs. The cDNA encoding the full-length wild- incubated with reductase at 377C for 5 min before the addition oftype coding sequence of CYP2D6 (40) was derived from the plasmid substrate. Incubations were carried out at 377C for 30 min andpMP201, which was constructed previously in our laboratory and stopped by addition of 5% (v/v) of 60% perchloric acid. The mixturesused elsewhere for the expression of CYP2D6 in yeast (20). The were centrifuged for 5 min and the supernatant was used for theCYP2D6 cDNA sequence in pMP201, flanked by EcoRI recognition determination of hydroxylated metabolites. Kinetic parameters forsites, extends from the ATG translation initiation codon to a position ({)-bufuralol 1*-hydroxylation were calculated from a Lineweaver–73 bp downstream of the translation termination codon in the 3* Burk plot. Metabolites were separated by HPLC on a ODS-2 5 mMnoncoding region. The entire 1567-bp CYP2D6 cDNA sequence was (Spherisorb) column using a Hewlet–Packard machine. 1*-Hydroxy-

bufuralol was detected with a fluoresence detector (excitation, 252nm; emission, 302 nm) and quantitated against known external stan-dards. Metoprolol assays were carried out using 1 mg protein in 1-2 Abbreviations used: Sf9, Spodoptera frugiperda; reductase,ml reactions containing 40 mM ({)-metoprolol and the generatingNADPH-cytochrome P450 reductase; PH, polyhedrin promoter; Pcor ,system was described. Metabolites were detected by fluorescence de-basic protein promoter; b2D6PH, recombinant baculovirus containingtection (excitation, 193 nm; emission, 280 nm). The reduction of cyto-CYP2D6 cDNA under the control of the polyhedrin promoter;chrome c by NADPH-cytochrome P450 reductase was carried out asb2D6cor , recombinant baculovirus containing CYP2D6 cDNA underpreviously described (17). One unit of reductase activity is definedthe control of the basic protein promoter; bR2D6, recombinant bacu-as the amount of enzyme which can reduce 1 nmol of cytochrome clovirus containing human NADPH-cytochrome P450 reductase andper minute.CYP2D6 cDNA’s; SDS–PAGE, sodium dodecyl sulfate–polyacryl-

amide gel electrophoresis; bRED, recombinant baculovirus con- Plate assay. In order to establish whether intact Sf9 cells car-rying the recombinant proteins could metabolize CYP2D6 substrates,taining NADPH-cytochrome P450 reductase cDNA; DTT, dithiothrei-

tol; EDTA, ethylene diaminetetraacetic acid, disodium salt; PBS, Sf9 cells were plated onto 24-well titer plates at a concentration of5.5 1 105 Sf9 cells per well. Cells were then infected with virus atphosphate-buffered saline.


145BACULOVIRAL EXPRESSION OF CYP2D6

crease in spectrally active P450 was also achieved us-ing the heme precursor d-amino levulinic acid (0.5 mM).To quantify the amount of active heme containing en-zyme, CO-reduced absorption spectra were measuredfrom detergent solubilized extracts (Fig. 2) or fromcrude cell lysates. The reduced CO difference spectrumwas typical of P450 proteins showing a peak maximumat 448 nm, with no sign of a peak at 420 nm; the specificcontent of CYP2D6 ranged between 100 and 200 pmol/mg of crude cell extract (0.5–1%) of total protein. Whengrown in suspension, Sf9 cells infected with b2D6PH

generally produced between 50–140 nmol CYP2D6/ perliter of cell suspension.

To determine whether the recombinant CYP2D6 wascatalytically active, cell lysates from Sf9 cells infectedwith b2D6PH were incubated with bufuralol and meto-FIG. 1. Time course of cytochrome P450 CYP2D6 production in Sf9prolol two prototypic CYP2D6 substrates. Purified ratcells. Sf9 cells infected with b2D6PH were grown in suspension andreductase was added (1000 Units/mg cell lysate) andremoved on the days indicated. Whole cells were solublised in sample

loading buffer (31 mM Tris–HCL, pH 6.8, 2% SDS, 10% glycerol, 100 both substrates were metabolized. Bufuralol 1*-hydrox-mM 2-mercaptoethanol, 0.002% bromophenol blue) and proteins were ylase activity was 0.9 nmol/min/nmol P450, which isseparated by SDS/10% PAGE. Each lane contains approximately 30 similar to levels of approximately 1.0 nmol/min/nmolmg total cell protein and kilodalton molecular weight markers (Mwt)

P450 reported for E. coli expressed CYP2D6 (27). Meto-are shown on the left.prolol can be attacked at two different positions by

a multiplicity of infection of 0.5 plaque forming units per cell and theplates were incubated at 277C with 1 ml substrate media consisting ofTNM-FH containing 2.5 mg/ml hemin and 10 mM ({)-bufuralol. Tomonitor enzyme activity, 150 ml of culture media was removed every4 h over a 3-day period. Culture media removed was replaced with150 ml of fresh media at each time interval. Perchloric acid (7.5 ml)was added to each sample removed and microfuged at 10,000g for 5min to precipitate proteinaceous material. To monitor the 1*-hy-droxy-bufuralol content, the supernatant fraction was subjected toHPLC analysis as described above.

RESULTS

CYP2D6 expression. CYP2D6 cDNA was clonedinto the transfer vector pVL1392 downstream of thevery late polyhedrin promoter (PH) and recombinantbaculovirus b2D6PH was generated by homologous re-combination with AcRP23.lacZ DNA. Following plaquepurification and virus amplification, infection of Sf9cells typically yielded very high levels of CYP2D6 pro-tein when visualized by SDS–PAGE with maximumlevels of CYP2D6 observed at around 3 days after infec-tion (Fig. 1). Baculoviral expressed CYP2D6 had thesame mobility as the native form found in human livermicrosomes. On solubilization with Emulgen 911, onlyapproximately 10% of the recombinant enzyme re-mained in the soluble fraction. Thus a large proportionof the baculoviral CYP2D6 was apparently an insolubleform. Recent purification of recombinant CYP2D6 indi- FIG. 2. Reduced carbon monoxide difference spectra of baculovirus

expressed CYP2D6. Sf9 cells infected with b2D6PH were pelleted andcates that most of the P450 in the soluble fraction issolublized with 1% Emulgen 911 in 50 mM potassium phosphate, pHin the form of active holoenzyme (34).7.5, 1 mM DTT, 1 mM EDTA, 20% glycerol (v/v), and the differenceAs previously reported, the presence of hemin chlo- spectrum was recorded using a supernatant fraction obtained follow-

ride in the growth medium was important for the pro- ing centrifugation at 38,000g for 30 min. Cells were grown in thepresence of 2.5 mg/ml hemin.duction of spectrally active enzyme (39). A similar in-


146 PAINE ET AL.

TABLE I

P450 and Reductase Contents in Sf9 Cell Extractsa

P450 Reductase activityBaculovirus (nmol/mg) (nmol/min/mg)

b2D6PH 0.15 { 0.03 —b2D6cor 0.17 { 0.01 —bR2D6 0.02 { 0.01 1266 { 118bRed — 1888 { 376

a Each value represents the mean { SD of three experiments.

CYP2D6 produced under the control of the basic pro-FIG. 3. Analysis of baculovirus expressed CYP2D6 and reductase. moter was generally less than that generated by theSDS/10% PAGE analysis was performed on whole cells infected with polyhedrin promoter, consistent with the weaker na-baculovirus and solubilized in sample loading buffer. Each lane con-

ture of the promoter element (Fig. 3). However, astains approximately 10 mg total cell protein. Lane 1, Sf9 cells infectedshown in Table I, Sf9 cells infected with b2D6cor pro-with b2D6PH; lane 2, uninfected Sf9 cells; lane 3, Sf9 cells infected

with bR2D6; lane 4, Sf9 cells infected with wild-type virus; lane 5, duced similar levels (0.17{ 0.01 nmol/mg) of spectrallySf9 cells infected with b2D6cor . Molecular weight markers are also active enzyme as CYP2D6PH (0.15 { 0.03 nmol/mg) .shown. To determine what proportion of the recombinant pro-

tein was expressed as soluble protein, Western blotanalysis with CYP2D6 antibodies of the detergent solu-

CYP2D6 to form O-desmethyl metoprolol and a-hy- ble and pellet fraction of infected cells was carried out.droxy metoprolol (19); both activities were observed in As shown in Fig. 4, the amount of insoluble apoproteinthe Sf9 cell extracts, with specific activities of 0.15 was significantly reduced when using the basic proteinnmol/min/nmol P450 and 0.06 nmol/min/nmol P450, re- promoter. Thus in comparison to the polyhedrin pro-spectively. Levels of enzyme activity were similar to moter, the basic promoter appears to provide a similaryeast expressed CYP2D6 activities of 0.2 nmol/min/ high yield of spectrally active CYP2D6 but with re-nmol P450 and 0.04 nmol/min/nmol P450 for O-des- duced levels of inactive apoprotein.methyl metoprolol and a-hydroxy metoprolol forma-

Coexpression of CYP 2D6 and NADPH cytochrometion, respectively (20). The ratio of O-desmethyl meto-P450 reductase. In view of the low endogenous activ-prolol:a-hydroxy metoprolol formed, 2.5, was some-ity of cytochrome P450 reductase in Sf9 cells, we deter-what lower than the ratio of 4.6 reported for CYP2D6

expressed in yeast (20).CYP2D6 expression under late promoter control. In

view of the high levels of apoprotein produced underthe control of the ‘‘very’’ late polyhedrin promoter wedetermined whether yields of active protein might beimproved through the use of an alternative promoter.The polyhedrin promoter is an extremely strong pro-moter (15) and it is possible that over production ofP450 protein coupled to its hydrophobic nature maylead to protein aggregation and incorrect folding. Fur-thermore the polyhedrin promoter is activated at a verylate stage of infection when there is a general suppres-sion of expression of Sf9 cellular proteins. This couldbe detrimental to the correct processing and folding of

FIG. 4. Western blot analysis of the distribution of cytochromeintegral membrane proteins. Thus a ‘‘late’’ promoter P450 CYP2D6 in Sf9 cells infected with b2D6PH and b2D6cor . Infectedsuch as the basic protein promoter (Pcor), which is cells were harvested, washed, and homogenized in 1% Emulgen inweaker and active at an earlier stage of infection, might 50 mM potassium phosphate, pH 7.5, 1 mM DTT, 1 mM EDTA, 20%

glycerol (10 mg cell protein/ml). Samples were then microfuged forbe more appropriate for CYP2D6 expression.10 min and approximately 10 mg of the solublized material (S) andUsing the transfer vector pAcMP2, CYP2D6 wasthe corresponding amount of insoluble protein (P) were subjected toplaced under the control of Pcor and the recombinant SDS/10% PAGE and Western blot analysis with CYP2D6 antibodies.

baculovirus b2D6cor was produced following homolo- Microsomes prepared from human liver (HLM) and yeast expressingrecombinant CYP2D6 (Y2D6) are shown.gous recombination. The total amount of recombinant



slightly increased by the inclusion of an external reduc-tase source—in marked contrast to the dramatic in-crease seen for b2D6PH system. The 1*-bufuralol hy-droxylase activity of CYP2D6 expressed by b2D6PH wasincreased over 20-fold by the addition of exogenous re-ductase. The fact that the low level of P450 reductasein Sf9 cells may make the enzyme rate limiting in incu-bations involving cell lysates is thus overcome throughthe coexpression of human reductase in these cells.

Kinetic constants for ({)-bufuralol metabolism wereestimated from Sf9 cells infected with bR2D6. The Vmax

value for bR2D6 of 0.37 nmol/min/mg was calculatedwith a turnover of 12.23 nmol/nmol P450/ min and aKm value of 4.7 mM. CYP2D6 activity was also stronglyinhibited by quinidine, a selective inhibitor of CYP2D6activity (30). The reported Ki of quinidine inhibition ofbufuralol hydroxylation in human liver microsomes byquinidine is 30 nM (31), and approximately 50% loss of

FIG. 5. Effect of exogenous P450 reductase on cytochrome P450 enzyme activity was seen at this concentration withCYP2D6 activity. Duplicate sample reactions containing 60 mg ofthe recombinant enzyme (Fig. 6).total cell protein from Sf9 cells infected with b2D6PH (j) and 128 mg

total cell protein from cells infected with bR2D6 (m) were incubated Plate assay. Heterologous expression systems in-with varying amounts of rat reductase in 175-ml reaction volumes cluding mammalian, yeast, and bacterial systems arecontaining 50 mM ({)bufuralol and a NADPH generating system (12.5 being increasingly used to determine the metabolic fatemM glucose-6-phosphate, 2.5 mM NADP, 10 units/ml glucose-6-phos-

of xenobiotic compounds. In this respect baculoviralphate dehydogenase). Incubations were carried out at 377C for 15min. expression is a potentially useful system since drug

interactions can be studied using infected whole cells,crude cell lysates, or microsome preparations. We were

mined whether activities could be improved and the interested to know whether the inclusion of enzymeneed to add reductase circumvented through coexpres- substrate during the course of the infection of Sf9 cellssion with human cytochrome P450 reductase. In order would enable metabolites to be monitored directly fromto do this CYP2D6 and human reductase were cloned the media.into the expression vector p2Bac under the respectivecontrol of the P10 and PH promoters, to produce bR2D6.Both foreign genes were expressed to a high level asdetected by SDS–PAGE gel analysis (Fig. 3).

As shown in Table I, the activity of human reductasein cells infected with bR2D6 and harvested 72 h afterinfection was in the range 1000–1500 units/mg andapproximately similar to that expressed in the absenceof P450. CO difference spectra of Sf9 cells infected withbR2D6 were generally difficult to detect in comparisonto b2D6 . The level of spectrally active coexpressedP450 was in the range 10–20 pmol/mg cell proteinwhich was significantly less than that expressed byb2D6PH or b2D6cor . A similar decrease in active P450content has been previously noted from Sf9 cells co-transfected with reductase and CYP2A1 (29), raisingthe possibility that high levels of reductase may havean adverse effect on P450 expression in Sf9 cells.

To determine if CYP2D6, when coexpressed withP450 reductase, could couple effectively to support drug FIG. 6. Effect of quinidine on bufuralol 1*-hydroxylation by bacu-

loviral expressed CYP2D6. Sample reactions containing 150 mg ofmetabolism, crude cell lysates from Sf9 cells infectedtotal protein from Sf9 cells infected with bR2D6 were incubatedwith bR2D6 were incubated with ({)-bufuralol in thewith varying concentrations of quinidine in 250-ml reaction volumespresence or absence of exogenous reductase. As shown containing 10 mM ({)bufuralol and an NADPH generating system.

in Fig. 5, coexpressed CYP2D6 and reductase enzymes Incubations were carried out for 30 min at 377C. 100% control activitywas 119 pmol/mg cell protein/min.appear to couple effectively since activity was only


148 PAINE ET AL.

and CYP3A4 (33), with highest levels of P450 expres-sion so far being achieved with CYP3A4 (0.46 nmol/mg total cell protein). In this study we have expressedCYP2D6 at high levels using the baculovirus system.Using the polyhedrin promoter, spectrally functionalCYP2D6 was expressed at levels of 0.075–0.17 nmol/mg cell protein and using suspension cultures we havebeen able to produce 100–150 nmol CYP2D6 per literof Sf9 cells. The enzyme represented around 1–2% oftotal cellular protein and the substrate specificity wasconsistent with native form in that it metabolized bu-furalol and metoprolol and was inhibited by quinidine.

To date, reported attempts to express P450’s in thebaculovirus system have exclusively employed thepolyhedrin promoter, since it is a strong promoter andconventionally used for the expression of heterologousFIG. 7. Time course of cytochrome P450 CYP2D6 activity in Sf9proteins in Sf9 cells. However, the strength of the pro-cells. Sf9 cells infected with bR2D6 (m), b2D6PH (j), and b2D6cor (l)

were incubated in growth media containing 10 mM ({)-bufuralol and moter coupled to the fact that activation coincides with1*-hydroxy bufuralol metabolite was measured by HPLC analysis a general suppression of cellular activity may be detri-at various times after infection as described under Materials and mental to the correct folding and heme incorporationMethods. Each point is the mean { SD of three separate determina-

and the proper processing of the membrane bound mol-tions.ecule. When expressed under the influence of theweaker late basic protein promoter we were able toproduce levels of active protein at least as high as the

Following infection of Sf9 cells with b2D6PH, b2D6cor , polyhedrin promoter, but with significantly reducedand bR2D6 in 24-well culture dishes, the presence of levels of insoluble apoprotein. Thus modulating expres-1*-hydroxy bufuralol metabolite in culture media sup- sion appears to provide a qualitative improvement inplemented with ({)-bufuralol was measured at various P450 yields in Sf9 cells. Tens of milligram quantities oftimes after infection (Fig. 7). Measurable amounts of purified protein are generally required for biophysical1*-hydroxy bufuralol were present in the media of all analysis by X-ray crystallography and NMR methods.cells expressing P450 2D6. There was no detectable We have recently shown that the expression systemactivity in cells infected with baculovirus which ex- described here, with the modification of the addition ofpressed human reductase alone (data not shown). In a ‘‘His-tag’’ to the protein to facilitate purification, canthe case of b2D6PH and b2D6cor , earliest enzyme activ- yield milligram quantities of pure soluble CYP2D6 fority was evident with cells infected with b2D6cor at NMR studies (34).around 28 h after infection, which is consistent with In addition to producing high levels of recombinantthe earlier activation of the ‘‘late’’ cor promoter. In com- protein for structure–function analysis, Sf9 cells alsoparison to b2D6PH, higher levels of activity were ob- provide a convenient system for the biochemical analy-tained with b2D6cor , which supports the view that ear- sis of P450-mediated substrate reactions as large quan-lier expression of CYP2D6, when the host machinery tities of Sf9 cells can be grown relatively easily andand endoplasmic reticulum is relatively intact, pro- infected cells can be used directly without the need forvides the optimal environment for the expression of membrane preparation procedures. A major drawbackactive enzyme. Overall, the highest activity levels were is the fact that the low level of P450 reductase in Sf9evident in cells infected with bR2D6. Taking into ac- cells may well make the enzyme rate limiting in P450count the fact that functional P450 levels are substan- reactions. It is thus necessary to add an exogenoustially lower using the coexpression system, this reflects source of reductase to incubations involving cell ly-efficient coupling of coexpressed human reductase and sates. This problem can be overcome by coinfection ofCYP2D6 enzymes. Optimal rates of metabolism were Sf9 cells with NADPH cytochrome P450 reductase (29)obtained 36 to 48 h after infection, correlating well with but requires extra cell culture manipulations. Wethe expected high level expression of the baculoviral therefore introduced both CYP2D6 and reductase into‘‘very late’’ promoters at this stage of the viral life the same transfer plasmid under the control of differentcycle (15). promoters and coexpressed the two components of the

monooxygenase system in Sf9 cells. Reductase was ex-DISCUSSION pressed at levels of up to 1500 units per mg of total cell

protein. It was notable that levels of spectrally activeThe baculovirus system has been used to express sev-eral P450 enzymes including CYP2A1 (39), CYP17 (32), CYP2D6 were significantly less when coexpressed with



reductase than when expressed alone. A similar phe- very late stage of infection, respectively. A qualitativeimprovement in the ratio of spectrally active to inactivenomenon has been noted when reductase was coex-

pressed with CYP1A2 (29) although not with CYP3A4 enzyme was achieved using the basic promoter and weare currently using this system in order to produce(28). We do not know the reason for this. However, it

has been demonstrated that oxidative damage of micro- recombinant CYP2D6 for NMR and X-ray crystallogra-phy studies. The optimization of catalytic activitysomal proteins can occur which is initiated by NADPH

and mediated by cytochrome P450 (25), and we are through coexpression of CYP2D6 and reductase in Sf9cells may also provide a system with which to charac-currently investigating whether supplementing the

growth media with antioxidants might prevent this oxi- terize the enzyme and its interaction with substratesat the molecular level.dative damage and increase levels of functional

CYP2D6.The reductase and CYP2D6 coupled effectively to ACKNOWLEDGMENTS

metabolize the CYP2D6 subsrate bufuralol withoutWe thank Brian McStay for advice regarding the use of the bacu-any need for an additional reductase being added. The

lovirus expression system. This work was supported by the Medicalrecombinant enzyme had an estimated kcat of 12.2 Research Council.min01 and a Km of 4.7 mM. These values correspondreasonably well with measurements of bufuralol 1*-hy-

REFERENCESdroxylation by CYP2D6 purified from human liver mi-crosomes (35–37) or from E. coli (27), for which kcat 1. Wolf, C. R. (1986) Trends Genet. 2, 209–214.values of 0.5 to 6.4 min01 and Km values of 19 to 60 mM 2. Nebert, D. W., Nelson, D. R., Coon, M. J., Estabrook, R. W.,

Feyereisen, R., Fuji-Kuriyama, Y., Gonzalez, F. J., Guengerich,have been reported. Recombinant CYP2D6 was alsoF. P., Gunsalus, I. C., Johnson, E. F., Loper, J. C., Sato, R.,inhibited by quinidine with an approximate 50% reduc-Waterman, M. R., and Waxman, D. J. (1991) DNA Cell Biol. 10,tion in enzyme activity occuring at the expected Ki of 301–14.

nM. Thus CYP2D6 expressed in Sf9 cells with human3. Eichelbaum, M., and Gross, A. S. (1990) Pharmacol. Ther. 46,reductase behaves in a functionally similar manner to 377–394.

the native enzyme. Similar coexpression of CYP3A4 4. Lennard, M. S. (1990) Pharmacol. Toxicol. 67, 273–283.and human reductase in insect cells has been reported

5. Ayesh, R., Idle J. R., Ritchie, J. C., Crothers, M. J., and Hetzel,recently (28), again exhibiting catalytic properties sim- M. R. (1984) Nature 312, 169–170.ilar to those found in human liver microsomes. Thus 6. Gough, A. C., Miles, J. S., Spurr, N. K., Moss, J. E., Gaedigk,reconstitution with purified reductase can be avoided A., Eichelbaum, M., and Wolf, C. R. (1990) Nature 347, 773–

776.and optimal catalytic activity can be obtained throughcoexpression of reductase using dual promoter transfer 7. Smith, C. A. D., Gough, A. C., Leigh, P. N., Summers, B. A.,

Harding, A. E., Maraganore, D. M., Sturman, S. G., Schapira,plasmids.A. H. V., Williams, A. C., Spurr, N. K., and Wolf, C. R. (1992)We were able to monitor CYP2D6 activity from in-Lancet 339, 1375–1377.fected Sf9 cells by adding ({)-bufuralol to the growth

8. Islam, S. A., Wolf, C. R., Lennard, M. S., and Sternberg,media and measuring 1*-hydroxy bufuralol metabolites M. J. E. (1991) Carcinogenesis 12, 2211–2219.directly from the media. Highest activity was obtained

9. Wolff, T., Distlerath, L. M., Worthington, M. T., Groopman,from cells coexpressing CYP2D6 and reductase, and J. D., Hammons, G. J., Kadlubar, F. F., Prough, R. A., Martin,the rate of activity was greatest between 36 to 48 h M. V., and Guengerich, F. P. (1985) Cancer Res. 45, 2116–2122.after infection. There are several advantages of this 10. Meyer, U. A., Gut, J., Kronbach, T., Skoda, C., Meier, U. T.,

Catin, T., and Dayer, P. (1986) Xenobiotica 16, 449–464.system for drug metabolism studies which include thefact that cofactor supplements are not required, there 11. Poulos, T. L., Finzel, B. C., Gunsalus, I. C., Wagner, G. C., and

Kraut, J. (1985) J. Biol. Chem. 260, 16122–16130.is minimal cell processing, thus minimizing enzyme12. Ravichandran, K. G., Boddapalli, S. S., Haseman, C. A.,instability, and assay procedures are simplified. We

Peterson, J. A., and Deisenhofer, J. (1993) Science 261, 731–have also been able to measure enzyme activity from736.Sf9 cells grown on a small scale using microtiter plates

13. Barnes, H. J., Arlotto, M. P., and Waterman, M. R. (1991) Proc.which facilitates the analysis of multiple samplesNatl. Acad. Sci. USA 88, 5597–5601.

allowing detailed kinetic analysis. Plate assay methods14. Smith, G. C. M., Tew, D. G., and Wolf, C. R. (1994) Proc. Natl.would also be ideally suitable for rapid screening of Acad. Sci. USA 91, 8710–8714.

inhibitors of cytochrome P450 activity, and also for 15. O’Reilly, D. R, Miller, L. K., and Luckow V. A. (1992) Baculovirusanalysis of slowly metabolized substrates. Functional Expression Vectors: A Laboratory Manual, Freeman, New York.analysis of CYP2D6 activity through site-directed mu- 16. Omura, T., and Sato, R. (1964) J. Biol. Chem. 239, 2370–2378.tagenesis studies might also be carried out. 17. Vermillion, J., and Coon, M. J. (1978) J. Biol. Chem. 253, 8812–

In conclusion, we have obtained high level expression 8819.of CYP2D6 in insect Sf9 cells using basic and polyhe- 18. Kronbach, T., Mathys, D., Gut, J., Catin, T., and Meyer, U. A.

(1987) Analyt. Biochem. 162, 24–32.drin promoters which are active at the late stage and


150 PAINE ET AL.

19. Otton, S. V., Crewe, H. K., Lennard, M. S., Tucker, G. T., and 31. Smith, D. A., and Jones, B. C. (1992) Biochem. Pharmacol. 44,2089–2098.Woods, H. F. (1988) J. Pharmacol. Exp. Ther. 247, 242–247.

32. Barnes, H. J., Jenkins, C. M., and Waterman, M. R. (1994) Arch.20. Ellis, S. W., Ching, M. S., Watson, P. F., Henderson, C. J., Sim-Biochem. Biophys. 315, 489–494.ula, A. P., Lennard, M. S., Tucker, G. T., and Woods, H. F. (1992)

Biochem. Pharmacol. 44, 617–620. 33. Buters, J. T. M., Korezekwa, K. R., Kunze, K. L., Omata, Y.,Hardwick, J. P., and Gonzalez, F. J. (1994) Drug Metab. Dispos.21. Penman, B. W., Reece, J., Smith, T., Yang, C. S., Gelboin, H. V.,22, 688–692.Gonzalez, F. J., and Crespi, C. L. (1993) Pharmacogenetics 3,

28–39. 34. Modi, S., Paine, M. J. I., Sutcliffe, M. L-Y., Primrose, W. U., Wolf,C. R., and Roberts, G. C. K. (1995) submitted for publication.22. Bradford, M. (1976) Anal. Biochem. 72, 248–254.

35. Gut, J., Catin, T., Dayer, P., Kronbach, T., Zanger, U., and23. Barbeau , A., Cloutier, T., Roy, M., Plasse, L., Paris, S., andMeyer, U. A. (1986) J. Biol. Chem. 261, 11734–11743.Poirer, J. (1987) Lancet 2, 1213–1216.

36. Schimada, T., Misono, K. S., and Guengerich, F. P. (1986) J.24. Poirer, J, Roy., M., Campanella, G., Cloutier, T., and Paris, S.Biol. Chem. 261, 909–921.(1987) Lancet 2, 386.

37. Schwab, G. E., Raucy, J. L., and Johnson, E. F. (1988) Mol.25. Mukhopadhyay, C. K., and Chatterjee, I. B. (1994) J. Biol. Chem. Pharmacol. 33, 493–499.269, 13390–13397.

38. Patten, C. J., and Koch, P. (1995) Arch. Biochem. Biophys. 317,26. Hasseman, C. A., Kurumbail, R. G., Boddupalli, S. S., Petersen, 504–513.

J. A., and Deisenhofer, J. (1995) Structure 3, 41–62.39. Asseffa, A., Smith, S. J., Nagata, K., Gillette, J., Gelboin, H. V.,

27. Gillam, E. M. J., Guo, Z., Martin, M. V., Jenkins, C. M., and and Gonzalez, F. J. (1989) Arch. Biochem. Biophys. 274, 481–Guengerich, P. F. (1995) Arch. Biochem. Biophys. 319, 540–550. 490.

28. Lee, C. A., Kadwell, S. H., Kost, T. A., and Serabjit-Singh, C. J. 40. Kimura, S., Umeno, M., Skoda R. C., Meyer U. A., and Gonzalez,(1995) Arch. Biochem. Biophys. 319, 157–167. F. J. (1989) Am. J. Hum. Genet. 45, 889–904.

29. Tamura, S., Korzekwa, K. R., Kimura, S., Gelboin, H. V., and 41. Henderson, C., and Wolff, C. R. (1992) in Methods in MolecularGonzalez, F. J. (1992) Arch. Biochem. Biophys. 293, 219–223. Biology, Vol 10: Immunochemical Protocols (Manson, M., Ed.),

The Human Press Inc., Totawa, NJ.30. Kobayashi, S., Murray, S., Watson, D., Sesardic, D., Davis,D. S., and Boobis, A. R. (1989) Biochem. Pharmacol. 38, 2795– 42. Cupp-Vickery, J. R., and Poulos, T. L. (1995) Nature Struct. Biol.

2, 144–153.2799.


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Functional High Level Expression of Cytochrome P450 CYP2D6 Using Baculoviral Expression Systems