Protein Expression and Purification 21, 303–309 (2001)doi:10.1006/prep.2000.1377, available online at http://www.idealibrary.com on

Overexpression and Purification of the Membrane-BoundCytochrome P450 2B4

A. Sami Saribas,*,1 Larry Gruenke,*,†,‡,§,2 and Lucy Waskell*,†,3

*Department of Anesthesiology, University of Michigan, Ann Arbor, Michigan 48108;†Department of Veteran Affairs Medical Center, Ann Arbor, Michigan 48105;‡Department of Anesthesia, University of California, San Francisco, California 94143; and

§Veterans Affairs Medical Center, San Francisco, California 94

Received September 7, 2000, and in revised form November 7, 2000

Expression of the membrane-bound cytochromeP450 2B4 by the pLW01-P450 expression vector, whichutilizes a T7 promoter, is markedly improved byemploying Escherichia coli strain C41(DE3) [Miroux,B., and Walker, J. (1996) J. Mol. Biol 260, 289–298; Brid-ges, A., Gruenke, L., Chang, Y.-T., Vasker, I., Loew, G.,and Waskell, L. (1998) J. Biol. Chem. 273, 17036–17049].Using this expression system, it was possible to rou-tinely obtain an average of 50–60 mg and as high as 100mg of cyt P450 2B4 per liter of cell culture in volumes of500 ml. An improved purification procedure for cytP450 2B4 is also described which allows recovery of30% of the expressed protein. It was possible in onestep using B-PER reagent and polyoxyethylene-9-lauryl ether to both lyse the E. coli and solubilize theexpressed cyt P450. Cyt P450 2B4 with a specific con-tent of 17 nmol/mg protein and a single band on poly-acrylamide gel electrophoresis was routinely isolated.The yield of cyt P450 from the improved purificationprocedure is twice that from the original procedure

and the purity of the recovered protein typically hasa specific content of 17 nmol cyt P450/mg of protein.q 2001 Academic Press

1 Current address: Neose Technologies Inc., 102 Witmer Road, Hor-sham, PA 19044.

2 Current address: Chiron, Emeryville, CA.3 To whom correspondence should be addressed: VA Medical Center,

Research 11R, 2215 Fuller Road, Ann Arbor, MI 48105. Fax: (734)213-6985. E-mail: waskell@umich.edu.

1046-5928/01 $35.00Copyright q 2001 by Academic PressAll rights of reproduction in any form reserved.

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Eukaryotic cytochromes P450 (cyts P450)4 are a su-perfamily of membrane-bound heme proteins which areinvolved in metabolizing a large variety of substratesfrom xenobiotics to steroids. Cyt P450 2B4 (CYP2B4),formerly known as cyt P450 LM2, was among the firstmammalian cyts P450 to be isolated in a highly purifiedstate. Because of its stability and ready availability, ithas long served as a model for studies of mammaliancyts P450. Haugen and Coon first described the purifi-cation of cyt P450 2B4 from phenobarbital-induced rab-bit liver in 1976 (1). The cyt P450 2B4 purified fromrabbits was subsequently found to be a mixture of cyto-chrome P450 2B4 and several other closely related iso-forms (2–5). Since mechanistic and structural studiesof cyt P450 rely on the availability of large quantitiesof pure cyt P450 isozymes, the expression of the cytP450 genes in bacterial systems is necessary to providea convenient source of the protein. There have beennumerous reports describing expression of human cytsP450 in bacterial cells (6–8). Richardson et al. (9) havereported the expression of several cyts P450 in the 2Csubfamily both from human and rabbit at high levelsin Escherichia coli after modifying the DNA sequenceof the N terminal membrane anchor. The expression ofcyt P450 2B4 in E. coli [JM109(DE3)pLysS] cells usingthe pLW01-P450 plasmid has previously been describedby (10). However, with this procedure the maximal ex-

pression of cyt P450 2B4 was 20–25 mg of protein perliter in 100-ml quantities. Recently, Miroux and Walker(11) reported the successful expression of membrane-bound and globular proteins using the E. coli strain

4 Abbreviations used: cyt P450, cytochrome P450; IPTG, isopropyl-b-D-thiogalactopyranoside.

303

304 SARIBAS, GRUENK

C41(DE3) which limits the accumulation of the mRNAof the expressed protein. This decreases the mRNA tox-icity and allows more of the target proteins to be ex-pressed. When these host cells were used in conjunctionwith a T7 expression vector (pET vectors, Novagen,WI), extraordinarily high levels (>800 mg/liter) of themembrane-bound heme protein cyt b5 were produced(12). Encouraged by our results with cyt b5, we haveemployed C41(DE3) cells for the expression of cyt P4502B4 as well. In this work, improvements are describedwhich allow expression of cyt P450 2B4 in larger quanti-ties with greater reliability using C41(DE3) cells, and

which allow recovery of a larger portion (30%) of the

streaked on a LB plate supplemented with 100 mg

expressed cyt P450 2B4 with a specific content of 17nmol/mg protein. These improvements have facilitatedthe isolation of large quantities of highly purified cytP450 2B4 for our mechanistic studies.

MATERIALS AND METHODS

Cell Line

C41(DE3) cells were kindly provided by Dr. J. Walkerof Cambridge University.

Materials

Sodium hydrosulfite (dithionite), d-aminolevulinicacid, carbenicillin, IPTG, polyoxyethylene-9-laurylether, Tergitol NP-10, dithiothreitol, deoxyribonucleaseI, lysozyme, cholic acid, Reactive Red 120 on beadedagarose (type 3000-CL), and octyl Sepharose CL-4Bwere purchased from Sigma. DE52 (diethylaminoethylcellulose) was purchased from Whatman. Bio-BeadsSM-2 were from Bio-Rad. The B-PER reagent (No.78248) was from Pierce Chemical Co. Hydroxyapatite(fast flow) was obtained from Calbiochem. All otherchemicals were reagent grade and were obtained fromcommercial sources. All spectra were recorded on a Var-ian Cary 300 spectrophotometer.

Aqueous stock solutions of 10% sodium cholate wereprepared from recrystallized cholic acid and aqueoussodium hydroxide. Stock solutions of 20% polyoxyethy-lene-9-lauryl ether in water were prepared fresh everyweek to avoid accumulation of peroxides. DE52 waspreequilibrated with 20 mM Tris acetate, pH 7.7, at48C before use and stored in buffer with 15% ethanol.

Miscellaneous Procedures

Total protein concentrations were determined by theBCA assay (Pierce Chemical Co.) using bovine serumalbumin as the standard. SDS–polyacrylamide gels(12%) were prepared and run according to Laemmli

(13). The gels were stained using Pierce Blue Codestain. Cyt P450 and cyt P420 content were determinedby the method of Omura and Sato (14) using an extinc-tion coefficient «450–490nm of 91 mM21 cm21. Cyt P420

E, AND WASKELL

concentrations were calculated using extinction coeffi-cients «420–490nm of 111 mM21 cm21 for cyt P420 and an«450–490nm of 241 mM21 cm21 for cyt P450. Since thehigh concentration of detergents used to solubilize theproteins interfered with the determination of cyt P450,appropriate controls with known amounts of the purecyt P450 and detergent were performed. This interfer-ence by added detergent may be secondary to light scat-tering by the detergent. To measure the concentrationof cyt P450 in the cell culture, 1 ml of cell culture wascentrifuged at 10,000 rpm for 1 min in an Eppendorfcentrifuge and the pellet was resuspended in 1 ml of a100 mM potassium phosphate buffer, pH 7.4, containing20% glycerol (v/v). Two hundred microliters of the cellsuspension were withdrawn and diluted with the samebuffer (1/4) and placed in a 1-ml masked quartz cuvettewith a Teflon stopper. The cuvette containing the cellsuspension was kept on ice and a small amount of solidsodium dithionite was added before mixing by inversionat least two times. The cuvette containing the cell sus-pension was then placed in a Cary 300 UV–Vis spectro-photometer and equilibrated at 158C. The baseline wasrecorded until no further change was observed (about15 min). Then carbon monoxide was gently blown onthe surface of the cell suspension for 30 s and the cuvettewas immediately closed with the Teflon stopper. Thesample was mixed by inverting the cuvette two timesand afterwards was placed back in the spectrophotome-ter. Scans were taken between 350 and 600 nm at 158Cuntil no further spectral change was observed (about15 min). Concentrations of cyt P450 and cyt P420 werecalculated from this difference spectrum as indicatedabove.

Expression of Cyt P450 in E. coli C41(DE3) Cells

The pLW01-P450 expression plasmid was con-structed as previously described (10). The pLW01-P450plasmid contains a high copy origin of replication fromCol E1 and the bacteriophage T7 promoter upstreamof the cyt P450 2B4 gene (15). Competent C41(DE3)cells, prepared using the calcium chloride method (16),were transformed with pLW01-P450.Colonies trans-formed with pLW01-P450 were selected on Luria Ber-tani (LB) agar plates supplemented with 100 mgcarbenicillin/ml. A single colony was inoculated into5 ml of LB liquid media supplemented with 100 mgcarbenicillin/ml and incubated with shaking overnightat 308C. The C41(DE3)/pLW01-P450 cells were storedin LB medium containing 15% glycerol at 2808C. Toexpress cyt P450 2B4 from stock culture, cells were

carbenicillin/ml and the plate was incubated at 308Covernight. The next morning a single colony was usedto inoculate 50 to 140 ml of LB medium supplementedwith 100 mg carbenicillin/ml. On the following day, 100

OVEREXPRESSION AND PURIFICA

ml of TB medium in a 500 ml Erlenmeyer flask wasinoculated with 2 ml of overnight culture or 500 ml ofTB medium in a 2.8-liter Fernbach flask was inoculatedwith 5 ml of overnight culture. Both flasks were supple-mented with 0.5 mM d-aminolevulinic acid and 100mg carbenicillin/ml. The cultures were grown at roomtemperature (22–238C) on an Innova orbital shaker at120 rpm overnight. After 20–22 h, when the cell densityreached an OD of 4–6 at 600 nm, cyt P450 2B4 expres-sion was induced by addition of IPTG (unless indicatedotherwise) to a final concentration of 0.7 mM. The cul-tures were grown for 94 h at room temperature withshaking at 120 rpm. At the end of the fourth day, 1-mlaliquots from each flask were withdrawn for determina-tion of the cyt P450 concentration. The cells from the1 ml aliquot were pelleted in an Eppendorf centrifugeat 10,000 rpm for 1 min, and either stored at 2308C orused immediately to determine the level of cyt P4502B4. The remaining cells were harvested by centrifuga-tion at 8000 rpm in a JA-10 Beckman Rotor at 48C. Thewet cell paste was placed in a 50-ml sterile culture tubeand stored at 2708C until purification. Typically 15–17g wet cell paste was obtained from 1 liter of cell cultureafter 4 days of growth.

Purification

Solubilization of cell pellets and chromatography onDEAE. Unless otherwise stated, all steps of the pro-tein purification were conducted at 48C. Cell pelletswere resuspended at 48C in 2.5 vol of 10 mM Tris acetatebuffer, pH 7.7, containing 1 mM EDTA, 0.1 mgdithiothreitol/ml, and 2% polyoxyethylene-9-laurylether. Cells were resedimented by centrifugation for 10min at 5g. Washed cell pellets (65 g) containing 50 nmolof cyt P450/g of cell paste were resuspended in a mixtureconsisting of 200 ml B-PER reagent, 50 ml of 20% poly-oxyethylene-9-lauryl ether, 20 mg dithiothreitol, 1 mgof deoxyribonuclease, and 40 mg of lysozyme. After themixture was frozen and thawed three times, sodiumcholate and glycerol were added to the resuspended cellpaste to final concentrations of 0.5 and 15%, respec-tively. The resulting mixture was applied to a 5 3 20-cm DE52 column which had been preequilibrated with20 mM Tris acetate, pH 7.7, at 48C, 20% glycerol, 0.1mM EDTA, 0.3% Tergitol NP-10 (buffer A). Cyt P4502B4 does not bind to the column and elutes in the voidvolume as the solubilized cell pellet is applied to thecolumn. The flow rate through the DE52 column shouldbe less than 1 ml/min to avoid bleedthrough of slowlybinding impurities. When all of the solubilized cell pel-let had been applied to the column, the remaining cyt

P450 was eluted with buffer A.

Chromatography on Reactive Red agarose. Frac-tions from the DEAE column with cyt P450 concentra-tions higher than 1 mM were pooled and applied directly

TION OF CYTOCHROME P450 2B4 305

to a 20.0-ml (1.5 3 11 cm) Reactive Red-agarose columnwhich had been preequilibrated with 20% glycerol, 0.1mM EDTA, 0.5% Tergitol NP-10 and 10 mM potassiumphosphate pH 7.4 (buffer B). The column was washedwith 15 column volumes of buffer B containing 20 mMpotassium phosphate. The cyt P450 was eluted withbuffer B containing 20 mM phosphate and 500 mMsodium chloride. As the cyt P450 began to elute a 1 310-cm Bio-Beads’ column which removed the bulk ofthe detergent was placed in the stream. The flow ratemust be kept below 0.5 ml/min to maintain the effi-ciency of the Bio-Beads’ column

Chromatography on octyl Sepharose and removal ofdetergent from the pure cyt P450. The fractions fromthe Bio-Beads’ column with a cyt P450 concentrationgreater than 3 mM were loaded directly onto a 45-ml(2.5 3 9 cm) octyl Sepharose column (30 nmol of cytP450/ml of resin) which had been equilibrated with 2vol of a solution of 200 mM potassium phosphate, pH7.7, 20% glycerol, 0.1 mM EDTA, 0.2% sodium cholate(buffer C). After the cyt P450 was loaded onto the col-umn, it was washed with one column volume of bufferC and 10 column volumes of 100 mM potassium phos-phate, pH 7.7, 20% glycerol, 0.1 mM EDTA, 0.4% so-dium cholate. The cyt P450 was eluted with a bufferwhich contained 100 mM potassium phosphate, pH 7.7,20% glycerol, 0.1 mM EDTA, 0.3% Tergitol NP-10. Thefractions from the octyl Sepharose column with cyt P450concentrations greater than 3 mM were combined anddialyzed overnight at 48C against 10 mM potassiumphosphate, pH 7.4, 20% glycerol, 0.3% Tergitol NP-10,0.1 mM EDTA. Tergitol is used in the dialysate to main-tain a similar osmolarity on each side of the dialysismembrane to prevent dilution of the cyt P450-con-taining solution. After dialysis, the solution was appliedto a 30-ml hydroxyapatite column (2.5 3 7 cm, 50 nmolcyt P450/ml hydroxyapatite) which had been equili-brated with 10 mM potassium phosphate, pH 7.4, 20%glycerol and 0.1 mM EDTA (buffer D). The column waswashed with this buffer until the absorbance at 280 nmof the effluent was no greater than 0.005 compared tothe applied buffer D. This normally requires washingwith 10–15 column volumes of the buffer. Cyt P450 waseluted with 400 mM potassium phosphate, pH 7.4, 20%glycerol, 0.1 mM EDTA.

The combined cyt P450 fractions were filteredthrough a 0.2-mm filter in order to sterilize the solutionand remove fine hydroxyapatite particles. The buffer

was then adjusted to 100 mM potassium phosphate (pH7.4) and 20% glycerol by dialysis. This solution wasconcentrated to give a final cytochrome P450 concentra-tion of between 100 and 200 mM and stored at 2308C.

306 SARIBAS, GRUENK

RESULTS AND DISCUSSION

Improvements in the Expression of Cyt P450 2B4 inE. coli C41(DE3)

The maximum cyt P450 2B4 expression was achievedwhen the cultures were induced with 0.7 mM IPTG atan OD600nm of 4.0 or higher and were grown for 94 h(total growth time 4 days) at room temperature withshaking at 120 rpm. If room temperature is not con-stant, the cultures should be grown in an incubatorfor maximum expression. Cultures of the wild-type cytP450 2B4 which had been incubated for 94 h expressedan average of 50–60 mg of the holoprotein per liter.Interestingly, the cyt P450 2B4 M137A mutant proteinwas expressed at slightly higher levels (85 mg/liter)than the wild type. The amount of cyt P450 2B4 ex-pressed after 94 h was significantly greater than thatobtained after 72 h of expression. IPTG concentrationswhich varied from 0 to 1.2 mM were tested and optimalexpression was observed with 0.7 mM IPTG, similar tothat reported by Miroux and Walker (11). In the absenceof IPTG, cyt P450 2B4 expression was less than 5 mgof holoprotein per liter of culture. The C41(DE3)cellstransformed with the pLW01 vector which lacks theCYP2B4 gene did not produce cyt P450. Temperaturewas an important factor in the expression of cyt P4502B4; maximum expression was achieved at ambienttemperatures (21–238C). At temperatures above 308C,the level of cyt P450 expression decreased while theexpression of the inactive form of cyt P450, cyt P420,increased. Another important factor was the cell den-sity at the time of induction with IPTG. Induction withIPTG was found optimal at cell densities above an ODof 4 at 600 nm. These induction conditions are similarto those used with JM109(DE3)pLysS host cells (10).Addition of the heme precursor d-aminolevulinic acid (d-ALA) to cultures increased the cyt P450 2B4 expression.The optimal d-ALA concentration was 0.5 mM. At lowerconcentrations cyt P450 expression was decreased. Athigher concentrations expression levels did not increasesignificantly. Richardson et al. (9) reported that theaddition of d-ALA to the growth medium increased theexpression of most but not all cyts P450 2C. It is notclear why the addition of d-ALA should affect the ex-pression of some cyt P450s but have no effect on theexpression of others.

After optimal levels of expression levels had beenachieved in 100-ml cultures, it was possible to scaleup the procedure and express cyt P450 2B4 in 500-mlcultures in 2.8-liter Fernbach flasks. Cyt P450 expres-sion typically ranged between 50 and 60 mg/liter. How-ever, levels as low as 30–40 mg or as high as 100 mg/

liter were observed in selected flasks. In the procedureused by Bridges et al. (10) with JM109(DE3)pLysS, typi-cal levels of cyt P450 2B4 expression were 20–25 mg/liter (450 nM) in 100 ml of medium. It was not possible

E, AND WASKELL

to achieve this level of expression in volumes greaterthan 100 ml. For example, in 2.8-liter Fernbach flaskscontaining 500 ml of medium the expression was only30–50% of that in 100-ml cultures. Thus C41(DE3) cellsnot only enabled higher levels of cyt P450 2B4 to beexpressed, but also allowed the expression to be con-ducted more efficiently in larger volumes. Another ad-vantage of the improved expression system is that itwas more consistent. The expression of cyt P450 2B4periodically failed in JM109(DE3)pLysS cells and cul-tures yielded little or no cyt P450. Presumably, thisoccurred because the expression vector was toxic and/oreasily lost in JM109(DE3)pLysS cells. It was thereforealways necessary to use freshly transformed cells toexpress the cyt P450 2B4. Since the pLW01-P450 vectoris stable in C41(DE3) cells, transformation of C41(DE3)cells with the plasmid DNA (pLW01-P450) before eachexpression is not necessary and overnight cultures forcyt P450 expression can be started from 15% glycerolstocks containing C41(DE3)/pLW01-P450 cells storedat 2808C. As described previously, cyt P450 2B4 is ex-pressed as the native protein. It was not necessary tomutate amino acids in the amino terminus to achievehigh expression levels (10).

Improvements in the Procedure for Purifying CytP450 2B4

Solubilization of cell pellets and chromatography onDEAE. The first step of the revised procedure bothlyses the E. coli and solubilizes the cyt P450. This is amarked advantage of the revised procedure and maybeapplicable to the purification of other membrane-boundcyts P450 expressed in E. coli. In the original procedure,cells were broken open by sonication. The “membrane”fraction containing the cyt P450 was then pelleted witha low-speed centrifugation. The cyt P450-containingpellet was resuspended and subsequently underwent ahigh-speed centrifugation at 100,000g. The pellet fromthe high-speed centrifugation was then resuspendedand solubilized. The soluble cyt P450 fraction was ob-tained following a second high-speed spin. Recovery ofcyt P450 from these steps was generally modest (lessthan 50%) and the specific cyt P450 content of the solu-bilized membrane fraction was typically low (>1 nmolcyt P450/mg protein). This was a problem, especiallywhen the expression was low, since the efficiency of theReactive Red–agarose column chromatography stepwas critically dependent on the specific content of cytP450 in the applied solution. In the modified procedure,the centrifugation steps are avoided by lysing the cellsand solubilizing the membrane proteins with deter-

gents in a single step. Although the revised purificationscheme requires an additional step of ion exchangechromatography on DEAE, the isolate after DEAE chro-matography has far greater specific content than the

After DEAE 510 1975 (110) 61 62 5Reactive Red and Bio-Beads 60 1265 (70) 39 62 12

Octyl Sepharose 85 1010Hydroxyapatite 25 1025

a 68 g of packed cells from 4 liters of cell culture.

solubilized membrane fraction from the original proce-dure. This results in greater reliability and higheryields from the Reactive Red column.

Certain details are critical for the success of the DE52column. If the rate at which the cyt P450 is loaded intothe column is too fast or the column is too short, theeffluent will become cloudy and the cyt P450 eluted willnot bind to the Reactive Red column. If the column is

weight standards; lane 2, C41(DE3)/pLW01-P450 crude cell extract.The C41(DE3)/pLW01-P450 cell paste was first boiled in SDS–polyacrylamide gel loading buffer. Then the soluble extract was di-rectly loaded onto the gel: Lane 3, purified cyt P450 2B4 (2.8 mg)after hydroxyapatite chromatography.

(56) 31 80 13(57) 31 100 17

whether DEAE–Sephacel or DEAE–Sepharose cansubstitute for DE52 in this purification protocol.

Reactive Red–agarose chromatography. The proce-dure for the Reactive Red–agarose chromatographystep is similar to that described previously (10), exceptas noted below. To maximize the yield from this stepthe size of this column should be adjusted to 1 ml ofresin for every 100 nmol of cyt P450 loaded onto thecolumn. This assures a recovery of at least 60% of theapplied cyt P450. In the current procedure, the columnis not washed with a buffer containing 100 mM sodiumchloride. To improve the purity of the final cyt P450preparation, a Bio-Beads’ column was placed in serieswith the Reactive Red column during elution of the cytP450. The Bio-Beads’ column removes the bulk of thedetergent with minimal effort. It is important to removemost of the detergent prior to chromatography withoctyl Sepharose since the detergent binds to the columnand displaces the cyt P450. The cyt P450 isolated fromthe Reactive Red column typically has a specific contentof about 11 nmol/mg. The specific content will be slightlyhigher after the Bio-Beads’ column. Note that detergentremoval with Bio-Beads is less efficient than detergentremoval on hydroxyapatite. Thus, the cyt P450 isolatedat this step contains detergent. In addition to removingthe detergent, the Bio-Beads’ column removes otherlow-molecular-weight impurities which bind to cyt P4502B4. This was evident from the fact that cyt P450 2B4isolated without use of the Bio-Beads’ column containeda greater amount of cyt P450 2B4 in the high-spin form.Thus, the Bio-Beads’ column should be used even if oneonly requires partially purified cyt P450.

Chromatography with octyl Sepharose and character-ization of the final product. The addition of an octylSepharose chromatography step to the original purifi-cation procedure yields a significant improvement inthe purity of the final product. The procedure for this

OVEREXPRESSION AND PURIFICATION OF CYTOCHROME P450 2B4 307

TABLE 1

Purification of Cyt P450 2B4 from E. coli

Cyt P450 % YieldCyt P450-specificcontent nmol/mg

Fraction Volume (ml) nmol (mg) overall step protein

E. coli a 3250 (181) 100 — —Lysed and solubilized cells 360 3200 (178) 98 98 0.3

long and thin, flow rates will be difficult to maintainas the column absorbs unwanted proteins from the solu-tion. These parameters should be considered carefullyin any attempt to rescale the procedure. It is not known

FIG. 1. SDS–polyacrylamide gel electrophoresis of cyt P450 2B4.A 12% gel was stained with GelCode blue stain. Lane 1, molecular

step is similar to that reported by Philpot and co-work-ers (17) except that sodium cholate is not included inthe elution buffer because it interferes with the removalof detergent by the subsequent hydroxyapatite column.

FIG. 2. (A) Absolute spectrum of 5 mM pure cyt P450 in 100 mMpotassium phosphate, pH 7.4, 20% glycerol at 258C. (B) The CO-bound minus reduced cyt P450 2B4 difference spectrum.

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Although sodium cholate is dialyzable, it is difficult toremove completely. It is important to remove all of thedetergent to obtain a pure product with consistent prop-erties. If one depends on dialysis to remove cholate, onewould need to use a specific and sensitive assay forcholate to determine when all of the cholate had beenremoved. Since detergents bind tightly to the protein,it is not clear for how long or against what volume onewould have to dialyze.

The procedure for removal of the detergent and purifi-cation on a hydroxyapatite column is unchanged fromthat used previously (10).

Characterization of the Purified Cyt P450 2B4

The yield and specific content of the protein aftereach step of the purification procedure are provided inTable 1. Analysis of the final product on a SDS–polyacrylamide gel showed a single band (Fig. 1). Anabsorbance spectrum of the protein taken between 240and 750 nm showed that the ratio between the Soretband at 418 nm and the absorbance at 277 nm was1.8 (Fig. 2A) which is consistent with the ratio of 1.9reported previously (17). The protein is isolated pre-dominantly in the low-spin form. The CO differencespectrum has a maximum at 450 nm and indicates thepresence of ,2% cyt P420 (Fig. 2B). In summary, themodified expression and purification procedures are amarked improvement over the old procedures. The cur-rent expression level is reproducibly 50–60 mg cytP450/liter versus 25 mg cyt P450/liter, while the overallyield of the purification procedure is now 30% versus15%. Although additional chromatographic steps are

required, the modified procedure is not significantly

308 SARIBAS, GRUENK

more work; the chromatography steps can be completedwithin 5 working days and the initial labor intensivesteps which involve centrifugation have been elimi-nated.

ACKNOWLEDGMENTS

We thank Ron Lee and Donna Jones for their help in growingcultures of E. coli. This work was supported by NIH Grant GM35533and a Department of Veteran Affairs Merit Review grant to L.W.

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TION OF CYTOCHROME P450 2B4 309

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