High-level P450

expression

human

cytochrome

1A2 in Escherichi#{225}coli

CHAR!IS ROBERT

of functional

W., FISHER, DEBORAH L CAUDLE, H. TUKEY$

MICHAEL

B. WAThRMAN,

MARTIN-WIXTROM, LINDA AND RONALD W. ESTABROOK’

CHERYL

C. QUATTBOCHI

tJnirsity of 1cas Southwestern Medical Center, Dallas, Texas USA; and Departmcnts and Medicine, University of California at San Diego Cancer Center, La Jolla, California USA

Departme#{252}t of Biochemistr)c

of Pharmacology

Enzymatically active human cytochrome P450 1A2 was expressed in Escherichia coil utilizing the pCWori + vector containing a modified cDNA. The coding sequence for the NH2-terminal region of the protein was modified by the alignment and substitution of a 27 bp segment from a modified bovine P450 17A1 cDNA onto the 5’ end of the open reading frame of P450 1A2 at amino acid 21. The expressed chimeric P450 was produced at a high level in a functionally intact form, as assayed by the formation in vivo of the 449 nm absorbance band of the CO complex of the reduced hemoprotein. E. coil membrane preparations were shown to contain P450 1A2, which was active in the 2-hydroxylation of estradiol, and the 0-deethylation of 7-ethoxycoumarin and 7-ethoxyresorufin, when reconstituted with recombinant rat liver NADPH-cytochrome P450 reductase. -Fisher, C. W.; Caudle, D. L.; Martin-Wixtrom, C.; Quattrochi, L. C.; Tukey, R. H.; Waterman, M. R.; Estabrook, R. W. High-level expression of functional human cytochrome P450 1A2 in Escherichia coil. FASEBJ. 6: 759-764; 1992. ABSTRACT

Key

ll7ords:

hydroxy/ation

cytochrome P450 . E. coli expression 1A2 0-dee! hylation

estradiol

UNDERSTANDING THE PROPERTIES OF cytochrome P450 1A2 is of great importance because it plays a critical role in the metabolism of 1) aromatic amines, 2) the natural food constituent caffeine, 3) the sex hormone estradiol, and 4) certain drugs including phenacetin, to name only a few substrates for this enzyme. The role of this P450 in catalyzing the N-oxidation of arylamines is considered a primary activation step for the formation of reactive metabolites that have carcinogenic potential (2, 3). Further, this P450 is of interest because it is induced in rats, and presumably humans, by agents such as polycyclic aromatic hydrocarbons, sidestream cigarette smoke, and heterocyclic amines formed during the charbroiling of meat (4). A variety of heterologous expression systems have been developed for the study of cytochrome P450s,2 including those of the 1A family. These systems include yeast (5-7), baculovirus (8), COS cells (3, 9), and vaccinia (2, 10-12) expression systems. All suffer from certain limitations including yield, ease of use, and expense. We have elected to use a bacterial expression system as it allows generation of large amounts of enzymatically competent enzyme in a relatively short time with minimal special requirements. Recent studies have produced functional P450s in Escherichia co/i, i.e., rabbit liver P450 2E1 (13), bovine adrenal gland P450 17A1 (14), and rat liver P450 7A (15). The applicability of the bacterial expression system to human liver P450s is of crucial

0892-6638/92/0006-0759/$01

.50. © FASEB

importance as it provides the ability to obtain sufficient quantities of material for enzymatic studies that would normally be quite limited. Many questions as to the function and structure of human liver P450s await to be answered. The production of human liver P450s in E. co/i, in a membrane devoid of any other P450s, will have many practical applications to the fields of toxicology and pharmacology. We report here the high-level expression of functional human P450 1A2 in E. co/i.

MATERIALS

AND

METHODS

Reagents Restriction enzymes Nde I and Xba I were obtained from New England Biolabs (Beverly, Mass.). T4 ligase, T4 kinase, and Klenow fragment of DNA polymerase were obtained from BRL (Bethesda, Md.). Taq polymerase was obtained from Perkin-Elmer Cetus (Norwalk, Conn.). Oligonucleotides were synthesized on an Applied Biosystems synthesizer (Foster City, Calif.). T7 polymerase sequencing kits were obtained from Pharmacia (Piscataway, N.J.). Protein molecular weight standards were from BioRad (San Francisco, Calif.). Isopropylthiogalactopyranoside (IPTG) was obtained from Research Organics, Inc. (Cleveland, Ohio). Recombinant rat liver NADPH-cytochrome P450 reductase was prepared from E. co/i containing the plasmid pOR263 as described (16). Activity of the purified reductase preparations ranged from 45 to 60 tmol cytochrome c reduced.min.mg protein. Estradiol (6,7[3H]) was obtained from New England Nuclear (Boston, Mass.) and repurified by chromatography on a celite partition column (17). DNA

and

plasmids

for human liver P450 1A2 was cloned from a human liver cDNA Xgtll library (18). The expression vector pCWori+ (19) was provided by Amy Roth, Department of Biochemistry, University of Oregon, and is similar to the previously described pHSe5 (20). The plasmid pTZ18R was obtained from Pharmacia, and competent E. co/i DH5cs cells were obtained from BRL.

A cDNA

‘To whom reprint of Biochemistry, 2Abbreviations:

cytochrome Nebert dodecyl

requests

should be addressed,

5323 Harry Hines The nomenclature

P450s

follows

et al. (1); PCR, sulfate; IPTG,

the

at: Department

Blvd., Dallas, TX and designation

recommendations

polymerase chain reaction, isopropylthiogalactopyranoside.

75235, USA. of various

described SDS,

by

sodium

759

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dithionite had been added, with the exception for in vivo assays where 2 mM glucose was also added to the bacterial Modifications to the 5’ and 3’ ends of the cDNA were persuspension. formed by polymerase chain reaction (PCR) amplification, The hydroxylation of estradiol was assayed using 3Hwith primers incorporating the desired sequences, which inlabeled estradiol essentially as described by Aoyama et al. cluded a Nde I site (CATATG) at the start of methionine and (12). Membrane preparations were diluted with a buffer mixan Xba I site 3’ to the termination codon. PCR conditions ture containing 50 mM Tris-HC1, pH 7.5, 150 mM KCI, were as follows: 20 mM Tris-HCI, pH 7.5, 50 mM KC1, 10 mM MgCl2, and 0.1 mM ascorbic acid to give 0.5 nmol 1.5 mM MgCls, 50 eM each dNTP, 1.25 U Taq polymerase of P450/ml. Purified recombinant rat liver NADPH-cytoin a 50 jzl reaction volume. The reaction mixture was cycled chrome P450 reductase was added to give a ratio of flavoin an MJR Research PCR machine (Cambridge, Mass.), protein to P450 of 2:1. The mixture was incubated at 37#{176}C temperature changes were made at maximum slope, i.e., for 10 mm, at which time radioactive steroid was added with 1#{176}C/mm.Cycling conditions were 94#{176}C, 30 5; 30#{176}C, 15 5; mixing to give the final concentration indicated (1-25 tM). and 72#{176}C, 30 s. The reaction was cycled 30 times and then One minute later the reaction was started by the addition of extended at 72#{176}C for 5 mm. The reaction product was exNADPH (0.3 mM final concentration) and a regenerating tracted with chloroform, filled in with the Klenow fragment system containing 4 mM sodium isocitrate and 0.3 units of of DNA polymerase I, and phosphorylated with T4 kinase. isocitrate dehydrogenase. Final volume of the reaction mixThe phosphorylated product was isolated on a low melting ture was 6 ml. Aliquots of 0.5 ml were removed at the times point agarose gel and extracted with phenol twice and chloindicated and added to 5 ml of dichloromethane, vigorously roform once. The PCR product was ligated into the Sma I mixed, extracted, and the organic phase was removed and site of pTZ18R and transformed into DH5cr cells. Recomevaporated to dryness with a stream of nitrogen. The sample binant colonies were screened by hybridization with 32p was dissolved in 100 tl of methanol:water (1:1) containing 1.5 labeled 5’ PCR oligonucleotide. The positive clones were semM ascorbic acid and immediately analyzed by HPLC. Aliquenced in their entirety with internal oligonucleotides to quots were automatically injected onto a C18 tBondapak detect any PCR errors. The Nde I-Xba I fragment was excolumn and metabolites were resolved using a 30-mm linear cised from the pTZI8R construct and ligated into pCWori +, gradient of 70:20:10 (water:methanol:acetonitrile) to 90:10 which had been digested with Nde I and Xba I. E. co/i DH5a (methanol:acetonitrile) using a Waters 840 HPLC system cells were transformed with the pCWori+ plasmid containconnected to a Radiometer Flo-l radiodetector. The acetoniing the human P450 1A2 insert. trile solution contained 1% acetic acid. The eluted products were assayed by tritium detector and their retention times Cell growth and harvesting were compared with known standards (12). 7-Ethoxyresorufin and 7-ethoxycoumarin 0-deethylase Conditions for the expression of P450 1A2 were modified activities were assayed fluorometrically using an Amincofrom those described by Barnes et al. (14) as follows: An overfluorometer as previously described (23, 24). Reacnight culture of the transformed E. co/i was grown at 37#{176}C Bowman tions were performed at 37#{176}C in a 2 ml volume using a in Luria-Bertani media containing 100 jg/ml ampicillin. A buffer consisting of 25 mM Tris-HCI pH 7.4, 150 mM KCI, 10 ml aliquot was used to inoculate 1 liter of Terrific broth 10 mM MgCls. Substrates were added as DMSO solutions (21) containing ampicillin, a mixture of trace elements (22), in less than 0.1% of the final volume. The reactions were inand 1 mM IPTG. The cells were grown in Fernbach flasks itiated by the addition of 10 el of 50 mM NADPH. for 72 h at 30#{176}C using gentle shaking (125 rpm). The cells were chilled on ice for I h, harvested by centrifugation at 3000 x g for 10 mm, and the pelleted cells were washed by RESULTS resuspending in one-fifth the volume using 10 mM potassium phosphate buffer, pH 7.5, containing 0.15 M NaCl and Modification of cDNA centrifuging at 12,000 x g for 10 mm. The supernatant was By aligning the amino acid sequences of the 5’ ends of bovine removed, and the pellet was drained and weighed. The cells P450 17A1 and several human P450 species we were able to were suspended using a Dounce homogenizer with a volume of TSE buffer (75 mM Tris-HC1, pH 7.5, 250 mM sucrose, detect a region of similarity. The alignment for human P450 1A2 with bovine P450 17A1 is shown in Fig. 1. We have and 0.25 mM EDTA) 2-fold the wet weight of cells. The found that constructions incorporating the NH2-terminal 9 suspended cells were divided into 100 ml aliquots and frozen amino acids from the modified bovine P450 17A1 produce at -70#{176}C. spectrally active P450s from several families of cytochrome For the preparation of membranes, cells were thawed and P450 (data not shown). This construction for human P450 subjected to two cycles of disruption with a cooled French 1A2, which incorporated a block of sequence from the 5’ end pressure cell. The disrupted cells were diluted with 2 volof the modified bovine P450 17A1 (Fig. 2), was found to exumes of TSE buffer and centrifuged at 3000 x g for 10 mm to remove unbroken cells. The supernatant was centrifuged press a competent chimeric P450 in E. co/i as determined by spectrophotometric assays. at 100,000 x g for 60 mm and the pellet was suspended in a minimal volume of TE buffer (50 mM Tris-HC1, pH 7.5, 0.5 mm EDTA), divided into aliquots, and stored frozen at 1. 10 20 MALSQSVPFSATELLLASAIFCLVF P450 1A2 -70#{176}C. Constructions

I I I I III

Enzymatic

P450

assays

Vol. 6

January

14WLLLAVFL 1

The level of expression of cytochrome P450 was determined spectrophotometrically with an Aminco DW2a Wavelength Scanning Spectrophotometer by measuring the magnitude of absorbance change at about 450 nm after addition of carbon monoxide to samples to which a few crystals of sodium

760

17A1

1992

9

Figure 1. Alignment of human P450 1A2 NH2-terminal amino acid sequence with the first nine amino acids of the amino terminus of bovine P450 17A1. Vertical lines represent identical residues, vertical dots represent conservative substitutions.

The FASEB Journal

FISHER ET AL.

.fasebj.org by Univ of Virginia Claude Moore Health Science Lib (128.143.7.175) on November 14, 2018. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueN

Bovine

P450

Human

17A].

2].

Met

Ala

Len

LCU

Len

Ala

ATO

OCT

CTO

TTh

TTA

OCA

Va]. OTT

Ph. TTT

P450

1A2

22

23

24

25

Lan Phe

Cys

Leu

Va].

Phe

TTC

TGC

CTG

GT

TTC

CTO

Figure 2. Construction used for expression of spectrally detectable human P450 1A2. Boldface letters indicate the block of sequence transferred from the modified bovine P450 hAl to the cDNA of P450

1A2.

Numbering

indicates

that

of the original

human

nm

449

E. coil Membranes

P450

Unit

1A2 sequence.

- Yield

of expressed

3

2

-

P450

Growth of cells in 1 liter volumes in Fernbach flasks showed an average level of expression of P450 1A2 of about 700 nmol of spectrophotometrically detectable P450/liter of growth media (about 12 g wet weight of cells). This indicates that about 4% of the total protein of E. co/i is represented by the holoprotein form of the recombinant P450. The recombinant P450 was stable in cells frozen at -20#{176}C for as long as 3 months. During disruption of the cells with the French pressure cell, approximately 50% of the spectrophotometrically detectable P450 was lost. However, the membrane fraction sedimented after centrifugation at 100,000 x g for I h had an average P450 content of about 2 nmol P450 per mg of protein (i.e., 10% of the membrane protein was P450). The high concentration of the expressed protein in this membrane fraction is shown by the sodium dodecyl sulfate (SDS) gel presented in Fig. 3. The appearance of the intensely stained band at about 52,000 Da, which is not seen in membranes from E. co/i containing the vector without an insert, supports this conclusion. Samples of purified rat P450 1A1 and P450 2B1 are included in this gel to show the similarity of electrophoretic mobility of recombinant human P450 1A2 to these rat forms. Also, the presence of a sample of an equal protein concentration of rat liver microsomes from f3-

1

of

4

5

6

7

8

-110 kD -84 kD -47

kD

Absorbance

Triton

I

I 400

U

U

U

U

I

‘

U

450 Wavelength

X100

I

Solubilized

I

500

(nm)

4. The spectrophotometric measurement of P450 lA2 bound to E. co/i membranes (A) and solubilized with 0.1% Triton X-100 (B). For A, membranes were diluted to 0.41 mg protein/mi Figure

in 0.1 M phosphate buffer, pH 7.4, and treated with sodium dithionite. After dividing the sample equally into two cuvettes, a baseline of equal light absorbance was recorded and then the contents of the sample cuvette were gassed with carbon monoxide for 1 mm. The unit of absorbance for A equals 0.05. For B, membranes were diluted to 2 mg protein/mi with phosphate buffer, pH 7.4, containing 10% glycerol. A 10% solution of Triton X-l00 was added dropwise to give a final concentration of 0.1%. The sample was centrifuged for 2 h at 100,000 x g. The supernatant was diluted with an equal volume of 0.1 M phosphate buffer, pH 7.4, to give a protein concentration of 0.3 mg/mI and the spectral changes were determined as described for A. The unit of absorbance for B equals

0.01.

-33 kO

Figure 3. SDS polyacrylamide gel electrophoresis of E. co/i membranes containing recombinant P450 1A2. Lanes I and 8 contain molecular weight standards (Bio Rad) of 110k, 84k, 47k, 33k Da; lane 3 shows the resolved proteins from 15 ig of membrane protein from E. co/i transformed with the pCWori + vector with a nonexpressed

insert;

lane 4 represents

E. co/i transformed

with

15 tg of membrane

the pCWori+

vector

containing

protein

from

the P450

1A2 modified cDNA; lanes 5 and 6 contain 1 g of purified rat P450 1A1 (form c) and P450 2Bl (form b), respectively; lane 7 shows the pattern

of proteins

associated

with

15 sg

of rat liver

microsomal

membranes from -naphthoflavone-treated rats. Results were obtained using a 10% polyacrylamide SDS gel. We are indebted to Dr. A. Parkinson for the samples of purified P450 1A1 and 2B1 shown

in lanes

5 and

6, respectively.

naphthoflavone-treated animals, as shown in lane 7, illustrates the comparatively higher level of expression of P450 1A2 in E. co/i compared with that obtained in the livers of induced animals. The amount of apoprotein of P450 1A2 cannot be estimated at this time because of the lack of an antibody specific for human P450 1A2 that can be used for quantitative titration. Recent preliminary studies indicate that about 40% of the P450 can be easily solubilized by addition of 0.1% Triton X-100 to membranes diluted to 2 mg protein/ml. (Solubilized is defined here as not sedimenting after 2 h of centrifugation at 100,000 x g.) The CO-difference spectra of the original membrane fraction and the Triton X-100 solubilized P450 are shown in Fig. 4, which illustrates the presence of little or no P420 in the material. The ability to solubilize P450 1A2 from the membrane by low concentrations of detergent offers the opportunity to purify large amounts of a human micro-

EXPRESSION OF RECOMBINANT HUMAN P450 1A2 761 .fasebj.org by Univ of Virginia Claude Moore Health Science Lib (128.143.7.175) on November 14, 2018. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueN

somal P450. These membranes will allow us to determine the influence of differences in protein and lipid composition of the membrane on the ability to solubilize a P450 from its membrane environment. Enzymatic

DISCUSSION The

alignment of a block of nine amino acids from the NH2sequence of bovine P450 17A1 shows a region of similarity with several different species of cytochrome P450. The presence of this similarity suggested that truncating the NH2-terminal sequence to align with the bovine l7Ah seterminal

TABLE 1. Kinetic parameters determined for reactions catalyzed membranes containing recombinant P450 IA?

by E. coli

Vmax,

Substrate

mol

min

-

Km,

mol P450-’

gM

Estradiol

1.5

13

7-Ethoxycoumarin 7-Ethoxyresorufin

0.36 2.5

44 0.01

were

determined

recombinant NADPH-P450

762

Vol. 6

E

assays

Membranes from E. co/i expressing P450 1A2 were incubated with the designated amounts of pure NADPH-cytochrome P450 reductase and the activity for the P450-directed oxidation of a number of substrates was determined. As shown in Table 1, recombinant P450 hA2 is active in the 0deethylation of 7-ethoxyresorufin and 7-ethoxycoumarin and the conversion of estradiol to the 2-OH metabolite (Fig. 5). Studies to determine additional enzymatic properties of the recombinant membrane-bound P450 1A2 have been carried out. Illustrated in Fig. 5 are the results of a series of experiments to determine the influence of varying the initial concentration of estradiol on the rate of formation of the 2and 4-hydroxylated products. Of interest is the observation that on/y 2-OH estradiol is formed during the first 5 mm of the reaction, when using membranes that had not been subjected to repeated freezing and thawing. Upon prolonged times (e.g., greater than 15 mm) of incubation of the reaction mixture, or when membranes were used that had been frozen and thawed several times, the formation of 4-OH estradiol and a more polar unknown metabolite was detected. These results suggest the instability of the metabolites formed or a modification of the properties of the P450 during prolonged incubation of the reaction mixture. Such an explanation may relate to the appearance of unknown metabolitesof estradiolas reported by Aoyama et al. (12). A second study has been carried out to determine the factors influencing the reaction of purified NADPH-cytochrome P450 reductase with the membrane-bound P450 hA2. As shown in Fig. 6, the influence of varying concentrations of flavoprotein reductase on the rate of 0-deethylation of 7-ethoxyresorufin, at a fixed concentration of membrane-bound P450 1A2, was determined. This experiment indicates the need for approximately 2.5 molecules of flavoprotein for each molecule of P450 in order to obtain the half-maximal rate of this reaction. Preliminary studies have indicated that this ratio depends on the composition of the reaction medium with a rather high concentration of salts in the medium required for highest activity.

‘Activities

C

Ianuarv

using

membrane-bound

P450 1A2 and

reductaseas describedin Methods.

1992

Th

AcFR

0.5 nmole 1A2J ml

10

0

+

1.0 nmole reduclase/mi

20

30

ESTRADIOL INITIAL CONCENTRA11ON (jsM)

0 C.,’

30 0

?2o

10

0.5

-0.5

1.0

1,5

l4iM ESTRADIOL

Km= 13 pAl Figure

5. The

hydroxylation of estradiol by membrane-bound

recombinant P450 1A2 reconstituted with recombinant rat liver NADPH-cytochrome P450 reductase. Membranes containing 1.5 nmoi of P450 IA2/mg were dilutedto 0.33 mg protein/mi using the buffer mixture described in Methods. A concentration of pure recombinant rat liver NADPH-cytochrome P450 reductase was added to give a ratio of reductase to P450 of 2 and the mixture incubated at 37#{176}C for hO mm. Varying concentrations of [3Hjestradiol were added as indicated and the reaction was started by addition of NADPH and a regenerating system. Final volume was 6 ml. Samples were taken at 1 mm intervals and the amount of 2-hydroxyestradiol formed was determined by HPLC analysis. of 2-hydroxyestradiol formed in 4 mm when using varying initialconcentrations of estradiol.B shows a reciprocal plot indicating a Km of 13 sM estradiol for obtaining the halfmaximal rate of the reaction.

A shows the amount

quence might allow expression of other P450s at levels seen with the modified bovine P450 17A1. By incorporating the 27 base pairs encoding for these nine amino acids onto the analogous region of the cDNA sequence, we are able to express high levelsof human P450 1A2, a form of P450 found in the microsomal fractionof human liver. The high levels of expression of P450 1A2 were not obtained with all P450 sequences modified in a similar manner (data not shown). This

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rIctlcp

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.fasebj.org by Univ of Virginia Claude Moore Health Science Lib (128.143.7.175) on November 14, 2018. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueN

2

1

0

2

4

6

8

10

ROdUCtaS&1A2(nmoUnmol) 6. The P450 reductase O-deethylation hA2 (40 pmol)

effect of varying the ratio of NADPH-cytochrome to membrane-bound P450 1A2 on the initial rate of of 7-ethoxyresorufin. A fixed concentration of P450 and 7-ethoxyresorufin (in 1 jil of DMSO, to a final concentration of 0.7 sM) was added to 2 ml of assay buffer (50 mM Tris-HCI, pH 7.5, 150 mM KCI, 10 mM MgCl2) with varying concentrations of NADPH-P450 reductase. The reaction mixture was equilibrated to 37#{176}C and initiated with 10 sl of 50 mM NADPH. The increase in fluorescence at 582 nm was monitored using an excitation wavelength of 560 nm. Figure

Enzymatically activehuman P450s, such as P450 1A2, can be used to predict what metabolites may be produced in vivo in humans (2, 9) and allow isolation of these metabolites in large quantity for structural determination. Current animal models for toxicology depend on the assumption that metabolites produced in the animal model are the same as those produced in humans. The ability to produce large quantities of human P450 metabolites will allow the direct testing of these metabolites in cell culture or other mutagenicity assays. For example, the catechol estrogens are proposed to play a role in the initiation of carcinogenesis. Their ability to undergo oxidation-reduction cycling, with the associated formation of radical intermediates (including superoxide), makes the understanding of their formation of central importance. One of the P450s active in the further metabolism of estrogens is P450 1A2. As shown in Fig. 5, the formation of 2-hydroxy estradiol is effectively catalyzed by the membranebound recombinant human P450 1A2, expressed in E. co/i, when reconstituted with purified rat liver NADPH-cytochrome P450 reductase. The initial rate of this reaction, using a ratio of P450 to flavoprotein reductase of 2:1, is about 1.5 nmol of estradiol metabolized per mm per nmol P450. This value is similar to that recently reported by Aoyama et al. (12), who studied the same reaction of P450 hA2 expressed in human Hep G2 cells transfected with recombinant vaccinia virus containing the cDNA for human P450 1A2. Our experiments indicate the instability of the hydroxylated products of estradiol metabolism. When the conversion of greater than 30-40% of the substrate occurs, a significant amount of water soluble metabolites is generated. In addition to answering basic questions concerning the enzymatic properties of these P450s, the ability to easily synthesize large amounts of active protein can now be exploited for commercial purposes as well. We wish to acknowledge

suggests that modification of the NH2-terminal segment of the protein was not the only factor responsible for this level of expression. Although it may be possible to customize the 5’ end of each P450 cDNA to optimize translational efficiency for that sequence, as was done for the expression of bovine P450 17A1, we believed it would be easier to use the 5 end of this modified bovine P450 17A1 (14) as a generic NH2-terminal sequence for the expression of other P450s, such as human P450 1A2. The use of this generic NH2terminal sequence maintains the regional secondary structural interactions of the RNA produced from the plasmid sequence. Modifications to the 5’ coding sequence have been found to increase the level of expression of several proteins in E. co/i (25-29). It is believed that the secondary structural interactions of the 5’ coding region and the region surrounding the ribosome binding site critically affect expression levels.In addition, this NH2-terminal amino acid sequence may also affect the correct folding, localization, and membrane insertion of cytochrome P450. In either case this procedure of alignment and substitution for expression has allowed the production of large amounts of active human P450 1A2 for structural and functional studies. Our results indicate that the NH2-terminal modifications do not interfere with the enzymatic activity of the recombinant membrane-bound form of human P450 1A2. Recombinant rat P450 7A, and recombinant rabbit P450 2E1 have both been expressed without the NH2-terminal membranebinding segment and yet still retain enzymatic activity (13, 15). Thus modifications at the NH2-terminal segment of the protein seems to be of little consequence to the expression of functional P450s.

Henry

J.

Barnes for helpful discussions

and the technical assistance of Margarita

Guijarro and Stephanie

Chatrein. We thank Dr. Charles Kasper, University of Wisconsin, for providing the plasmid p0R263 used for the preparation of

NADPH-cytochrome

P450

reductase

ported in part by a Sponsored University

of Texas

Southwestern

(16). This

work

Research Agreement Medical

Center

was sup-

between at Dallas

the and

Dallas Biomedical Corporation and by a grant from the National Institutes of Health (NIGMS 16488, RHT, GM36590). The procedures described in this paper are the subject of a pending

patent

(RB 184 695 522).

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liver cytochrome DNA 4, 203-210

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7. Cullin,

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Pompon,

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T., Yamano,

S., Guzelian,

P. S., Gelboin,

H. V., and

Gonzalez, F. J. (1990) Five of 12 forms of vaccinia virusexpressed human hepatic cytochrome P450 metabolically activate aflatoxin B. Proc. NaIL Acad. Sci. USA 87, 4790-4793 12. Aoyama, T., Korzekwa, K., Nagata, K., Gillette, J., Gelboin, H. V., and Gonzalez, F J. (1990) Estradiol metabolism by com-

plementary 13.

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The FASEB lournal

Received for publicalion September 30, 1991. Accepted for publication October 26, 1991.

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