Proc. Natl. Acad. Sci. USA Vol. 88, pp. 5597-5601, July 1991 Biochemistry
Expression and enzymatic activity of recombinant cytochrome P450 17a-hydroxylase in Escherichia coli (steroid hydroxylase/bacterial expression/reductase)
HENRY J. BARNES, MICHAEL P. ARLOTTO,
AND
MICHAEL R. WATERMAN*
Department of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235
Communicated by Ronald W. Estabrook, March 11, 1991 (received for review February 6, 1991)
When the cDNA encoding bovine microsomal ABSTRACT 17a-hydroxylase cytochrome P450 (P45017a) containing modifications within the first seven codons which favor expression in Escherichia coli is placed in a highly regulated tac promoter expression plasmid, as much as 16 mg of spectrally detectable P45017a per liter of culture can be synthesized and integrated into E. coli membranes. The known enzymatic activities of bovine P45017a can be reconstituted by addition of purified rat liver NADPH-cytochrome P450 reductase to isolated E. coli membrane fractions containing the recombinant P45017a enzyme. Surprisingly, it is found that E. coli contain an electrontransport system that can substitute for the mammalian microsomal NADPH-cytochrome P450 reductase in supporting both the 17a-hydroxylase and 17,20-lyase activities of P45017a. Thus, not only can E. coli express this eukaryotic membrane protein at relatively high levels, but as evidenced by metabolism of steroids added directly to the cells, the enzyme is catalytically active in vivo. These studies establish E. coli as an efficacious heterologous expression system for structurefunction analysis of the cytochrome P450 system.
Microsomal cytochromes P450 are integral membrane hemoproteins that catalyze the oxidative metabolism of a wide variety of endogenous and exogenous compounds. Deriving reducing equivalents from NADPH via a membrane-bound flavoprotein oxidoreductase (NADPH-cytochrome P450 reductase), these mixed-function oxidases activate molecular oxygen so as to insert one atom into a lipophilic substrate and the other atom into water. Recent study of the molecular aspects underlying eukaryotic cytochrome P450 structure and function has relied on the techniques of molecular biology to synthesize specific individual forms of cytochrome P450 in heterologous expression systems. Yeast (1), COS 1 (2), and eukaryotic cells infected with a viral vector (3, 4) have been used as hosts for the heterologous expression of cytochrome P450 molecules; however, each has limitations to their usefulness as systems for structure-function analysis. Although the bacterium Escherichia coli has demonstrated great usefulness in the expression of many prokaryotic and eukaryotic proteins, E. coli as an expression system for cytochrome P450 has been limited primarily to the soluble prokaryotic forms of this gene superfamily (5). We have used the cDNA encoding bovine 17a-hydroxylase cytochrome P450 (P45017a) to examine the utility of E. coli as an expression system for eukaryotic cytochromes P450 in the hopes that such a system might prove suitable for both enzymatic and structural studies. This microsomal cytochrome P450 catalyzes the regiospecific and stereospecific 17a-hydroxylation of the C21 steroids pregnenolone and progesterone in the pathway leading to the production of cortisol and the 17,20-lyase conversion of 17a-hydroxypreg-
nenolone to the C19 adrenal androgen dehydroepiandrosterone (DHEA) in the adrenal cortex of most mammalian species. P45017a also converts these 17a-hydroxylated products to the C19-androgen precursors of sex hormones via the 17,20-lyase reaction in the gonads of all species of mammals. P45017a [product (CYP17 in ref. 6) of the CYPJ7 gene (6)] is a typical representative of the large number of microsomal cytochrome P450 enzymes, and it is expected that knowledge gained in the study of bacterial expression of this specific cytochrome P450 will be generally applicable to this larger group.
MATERIALS AND METHODS Bacterial Strains and Plasmids. The E. coli strains used in this work were JM109 (7) and AT713 [A-, cysJ, argA21, lysA22, rpsL104, malAJ(AR), xyl-7, mtl-2] (8). The plasmid pCWOri+, a derivative of pHSe5 (9, 10), was used to express wild-type or modified P45017a cDNA sequences. This plasmid contains two tac promoter cassettes (Pharmacia no. 27-4883-01) upstream of an Nde I (CATATG) restriction enzyme cloning site coincident with the initiation ATG codon. The vector also contains a strong trpA transcription terminator sequence, a phage M13 origin of DNA replication, and the lacIq gene encoding the Lac repressor molecule that prevents transcription from the tac promoters prior to addition of inducing agents. Native and modified cDNA sequences were introduced into the expression plasmid pCWOri+ via polymerase chain reaction (PCR) mutagenesis (11). Synthetic oligonucleotides containing the native or mutant 5' cDNA sequences were used in conjunction with a downstream oligonucleotide to amplify the sequences between the ATG initiator codon (contained within an Nde I site) and a unique EcoRI restriction site of the plasmid pCD17a-2 (12). Following subcloning and sequencing of the amplification products, the expression plasmids were constructed by the simultaneous ligation of Nde I/HindIIIcleaved vector DNA with a 1257-base-pair (bp) EcoRI/ HindIll DNA fragment containing the cDNA encoding P45017a amino acids 92-509 and a 272-bp PCR Nde I/EcoRI fragment containing the native or modified cDNA PCR fragments encoding P45017a amino acids 1-91. The final expression plasmids (pCWnatl7 and pCWmodl7) were subjected to diagnostic restriction enzyme analysis prior to transformation into E. coli. Bacterial Expression and Cellular Fractionation. Ampicillin-resistant colonies of JM109 cells transformed with plasmid DNA were streaked on a fresh Luria-Bertani (LB) agar/ampicillin plate (10 g of tryptone, 5 g of yeast extract, 5 g of NaCl, 100 mg of ampicillin, and 15 g of agar per liter) Abbreviations: PCR, polymerase chain reaction; IPTG, isopropyl
/-D-thiogalactopyranoside; P45017a, 17a-hydroxylase cytochrome
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
P450 (also called CYP17), the product of the CYP17 gene; DHEA, dehydroepiandrosterone. *To whom reprint requests should be addressed. 5597
5598
Biochemistry: Barnes et al.
and were grown overnight at 370C. A single isolated colony was placed into LB/ampicillin medium and grown at 370C with shaking to near saturation. Sterile glycerol was added to this culture to a final concentration of 15% and aliquots were stored frozen at -70TC. These frozen samples served as innoculum for further expression experiments. Briefly, E. coli harboring the P45017a expression plasmids were grown to an OD550 = 0.4-0.8 in TB broth (13) containing 50-100 pug of ampicillin per ml at 370C. Where indicated, induction of the tac promoters was initiated by 1 mM isopropyl 8-Dthiogalactopyranoside (IPTG). The cells were shifted to 280C and gently shaken for 48 hr, pelleted, washed once in 50 mM 3-(N-morpholino)propanesulfonic acid (Mops) buffer (pH 7.5) containing 100 mM KCI, 1 mM EDTA, and 1 mM dithiothreitol and were resuspended in the same buffer (1/ 20th of the original culture volume). Lysozyme was added to a final concentration of 0.2 mg/ml, and the cells were incubated on ice for 30 min. Phenylmethylsulfonyl fluoride (100 mM stock in isopropanol), leupeptin, and aprotinin were added to final concentrations of 1 mM, 0.1 tug/ml, and 0.04 units/ml, respectively. The resulting spheroplasts were transferred to (2 x 4) cm glass vials placed in salt ice (-50C) and lysed by three 20-sec pulses from a 1.2-cm diameter probe attached to an Artek Sonic Dismembrator (model 150; Artek Systems, Farmington, NY) at 60% ofmaximum power. Unbroken cells and debris were pelleted at 1200 x g for 10 min. MgCl2 (6 mM) was added to the supernatant, which was centrifuged at 225,000 x g for 30 min at 4°C (14). The resultant membrane pellet was resuspended in Mops buffer containing 6 mM MgCl2 by gentle homogenization and was recentrifuged at 225,000 x g as before. After centrifugation, this washed membrane pellet was resuspended in Mops buffer. For electrophoretic analysis, a total cell protein fraction was prepared by boiling cells in 62.5 mM Tris HCl (pH 6.8) containing 2% SDS. Samples were fractionated on SDS/8% polyacrylamide gels and transferred to nitrocellulose membranes for immunoblot analysis (15). Spectral Studies. To obtain a P450 difference spectrum (23) in whole E. coli cells, cells treated with the reducing agent sodium dithonite and bubbled with CO were contained in the sample cuvette and cells treated with reducing agent alone were contained in the reference cuvette (reduced CO/reduced difference spectrum). A 100-ml culture of strain JM109 harboring pCWmodl7 was grown and induced as described above. Cells were washed and resuspended in 5 ml of Mops buffer. A 0.5-ml aliquot of concentrated cells was diluted with 5.5 ml of Mops buffer containing 10 mM glucose and was divided equally between two cuvettes. Several grains of sodium dithionite were added to each cuvette, and the baseline reduced spectrum was recorded in an Aminco DW-2A spectrophotometer. CO was then bubbled through the sample cuvette, and the reduced CO/reduced difference spectrum was recorded. For substrate binding spectra, a volume of washed cells containing 1.6 nmol of P45017a was diluted to 6 ml with Mops buffer and divided into the sample and reference cuvettes. A baseline tracing was recorded from 350 to 510 nm. Steroid was added in 10-,l aliquots (1-2 mg/ml in ethanol) to the sample with an equal volume of ethanol added to the reference. Additional aliquots of steroids and ethanol were added until the bacterially expressed P45017a was saturated as determined spectrally. Steroid Metabolism of the Recombinant Enzyme. For in vivo metabolism, E. coli were cultured and induced as described above. Cells were pelleted, washed once in Mops buffer, and resuspended in the same buffer at 1/20th of the volume of the original bacterial culture. A volume of concentrated cells corresponding to 3.7-4.2 nmol of P45017a was diluted to S ml in Mops buffer containing 10 mM glucose, 2.5 AM steroid, and 100,000 cpm of 3H-labeled radioactive steroid per ml. Samples were incubated at 280C with gentle shaking. Aliquots
Proc. Natl. Acad. Sci. USA 88 (1991)
(0.5 ml) were removed at 0, 0.5, 1, 2, 4, 6, and 8 hr after steroid addition. After extraction of cells and culture media, steroids were analyzed by HPLC as described (16). For in vitro reconstitution of enzyme activity, E. coli membrane fractions were prepared as described above (14) with the following modifications. Potassium glutamate buffer [KGB; 25 mM Tris acetate, pH 7.6/10 mM Mg(OAc)2/100 mM L-glutamic acid, monopotassium salt/0.1 mM dithiothreitol] (17) containing 10% (wt/vol) glycerol replaced Mops buffer for washing and sonication of cells. Washed cell pellets were resuspended in this buffer at a ratio of 2 ml ofbuffer per g (wet weight) of cells. A soluble protein fraction (2.9 ml/g of cells) was obtained by treating cells with lysozyme and sonication, followed by low-speed removal of debris and high-speed pelleting of E. coli membranes. This soluble fraction was dialyzed overnight at 40C against three changes of 300 ml of KGB/glycerol buffer. The dialyzed protein solution was centrifuged at 225,000 x g for 30 min to remove aggregated material, and the supernatant (now 2.4 ml/g of cells) was used as the cytosol fraction. The membrane pellet was resuspended in 0.25 M sucrose. Both cytosol and membrane fractions were stored at -70'C until use. For reconstitution of P45017a activities, membranes containing 0.5 nmol P45017a were combined with different amounts of purified rat liver microsomal NADPH-cytochrome P450 reductase (18) or with dialyzed E. coli cytosol at 370C. The P45017a/ cytosol ratio was maintained at 1:1 based on the cell mass used for initial fractionation. After a 10-min preincubation, reactions were initiated by addition of NADPH (1 mM) and steroids (5 AM plus 105 cpm of 3H-labeled radioactive steroid per ml) in a final volume of one ml of KGB or 50 mM potassium phosphate buffer (pH 7.5), each containing 12% glycerol. Samples were shaken vigorously at 370C, 200-gl aliquots were removed at various times after NADPH/ steroid addition and extracted immediately with methylene chloride, and steroids were analyzed by HPLC.
RESULTS AND DISCUSSION For expression in bacteria, the cDNA for the coding region of bovine P45017a (12) was cloned into the E. coli expression vector pCWOri+. Upon transformation of this expression plasmid containing the native codons of P45017a into the E. coli strain JM109, no immunoreactive P45017a protein was produced following derepression of the tac promoters (Fig. 1B, lane 2). Examination of the amino-terminal coding sequence of P45017a, based on the reports by others that this region plays an important role on expression levels in E. coli, led to alterations within the first seven codons of the cDNA by PCR mutagenesis as indicated in Fig. 1A in an attempt to optimize parameters for bacterial expression. Specifically, the native second codon was changed from TGG (Trp) to GCT (Ala), a preferred second codon for expression of the lacZ gene (19), and codons 4 and 5 were changed to TTA (silent mutations), since this region of E. coli mRNAs has been shown to be rich in adenosine and uridine nucleotides (20). Also the last nucleotide of codons 6 and 7 was changed to adenosine and thymidine (silent mutations), respectively, to minimize secondary structure formation in the messenger RNA (21). Immunoblot analysis (Fig. 1B) indicates that these alterations have a profound effect on the expression of P45017a in E. coli and that this expression is efficiently repressed in the absence of IPTG. Fractionation of transformed E. coli into membranes and cytosol (14) established that the expressed P45017a is associated with the membranes. Furthermore, the P45017a enzyme present in these membranes is not rendered soluble by treatment with 0.1 M sodium carbonate (pH 11.5), indicating that it is an integral membrane protein (22) in E. coli (data not shown). Only very
Biochemistry: Barnes et al.
Proc. Natl. Acad. Sci. USA 88 (1991)
teristic 450-nm absorbance maximum of all cytochromes P450 (23). Binding of substrates was also observed by detection of substrate-induced difference spectra (24) in intact bacteria after addition of the C21 steroids pregnenolone, progesterone, 17a-hydroxypregnenolone, or 17a-hydroxyprogesterone (Fig. 2B). The C19 steroid product of the bovine 17,20-lyase reaction, DHEA (16), showed much less binding, whereas another C19 steroid, androstenedione, which is not a product of bovine 17,20-lyase (16), showed no binding. The functional role of the binding of C21 steroids to the expressed P45017a was determined by incubation of transformed E. coli membranes with purified rat liver NADPH-cytochrome P450 reductase and radiolabeled substrates in a reconstitution assay followed by HPLC analysis of substrates and products. The 17a-hydroxylase and 17,20lyase activities known to be associated with bovine P45017a could be observed in reconstituted systems (Fig. 3). The turnover number of about 1.0 min-' for conversion of progesterone to 17a-hydroxyprogesterone by bovine P45017a in E. coli membranes (Table 1) is in the same range but smaller than that of 6.9 min- reported for purified pig P45017a (25). The moderate difference in turnover numbers may be due to intrinsic differences between species and/or failure to achieve maximal reconstitution of activity in bacterial membranes. Surprisingly, E. coli are capable of supporting the enzymatic activities of P45017a. The enzymatic profile (Fig. 4) of the expressed bovine P45017a in bacteria was the same as that observed in other heterologous expression systems, COS-1 cells (16) and yeast (27). Briefly, pregnenolone and progesterone were readily converted to their 17a-hydroxylated products, and 17a-hydroxypregnenolone was converted to the C19 steroid DHEA, whereas 17a-hydroxyprogesterone was not converted to androstenedione. Furthermore, when pregnenolone was added as substrate it first was converted almost entirely to 17a-hydroxypregnenolone be-
A nat17
ATG TGG CTG CTC CTG GCT GTC TTT Met Trp Leu Leu Leu Ala Val Phe
modl 7
ATG GCT CTG TTA TTA GCA GTT TTT Met Ala Leu Leu Leu Ala Val Phe
B
1
2 3 4 5 6 7 ...:..
...
17aH, l~~~~aOH'~ ~ ,,,
;
^
FIG. 1. Nucleotide and amino acid sequences at the 5'-ends of native (natl7) and modified (modl7) P45017a cDNAs and their expression in E. coli. (A) The nucleotide changes (indicated in boldface letters) introduced via PCR mutagenesis. (B) Immunoblot analysis of bacterially expressed 17a hydroxylase (17aOH). Lanes: 1, 9.4 Mg of bovine adrenocortical microsomes; 2, 50Mug of total cell protein (TCP) of strain JM109 (pCWnatl7) + IPTG; 3, 50 tg of TCP JM109 (pCWmodl7) - IPTG; 4, 50 Mg of TCP JM109 (pCWmodl7) + IPTG; 5, a mixture of samples shown in lanes 6 and 7; 6 and 7, the 225,000 x g supernatant and pellet fractions, respectively, from a 200-Ml culture of JM109 (pCWmodl7) + IPTOG.
minor proteolytic degradation of P45017a is evidenced by immunoblot analysis. Not only is the P45017a protein expressed in E. coli, but also this protein has the spectral characteristics of functional P45017a (Fig. 2j. The reduced CO/reduced difference spectrum obtained in intact E. coli (Fig. 2A) shows the charac-
A
5599
B Preg 17caOH-Preg
1~~~~~~~~~~0.01
0
DHEA
0.01
Baseline
370
a -0
390
410
430
450
Baseline
co Qa
Co
0
-0
E1 7aOH-Prog
.0~~~~~~~~~~00
0
AD _____________________________________
420
450
Wavelength (nm)
480
370
__
Baseline
390 410 430 450 Wavelength (nm)
FIG. 2. Reduced CO/reduced difference spectrum (A) and substrate binding spectra (B) of P45017a in intact E. coli cells. Steroids and their final concentrations in B are pregnenolone (Preg), 22MLM; 17a-hydroxypregnenolone (17&OH-preg), 40MAM; DHEA, 46MLM; progesterone (Prog), 21 MM; 17a-hydroxyprogesterone (17aOH-prog), 40 MM; and androstenedione (AD), 46 uM.
5600
2\/~
Proc. Natl. Acad. Sci. USA 88 (1991)
Biochemistry: Barnes et al.
4--
0 0
._
0o
0
30
1 5
45
60
2
0
Incubation, min
4
6
8
1 0
Incubation, min
FIG. 3. Steroid metabolism of isolated E. coli membranes supplemented with purified rat liver NADPH-cytochrome P450 reductase. Reactions were performed in KGB/glycerol buffer at a reductase/P45017a molar ratio of 1:1. Initial substrate concentrations were 5 ,uM.
fore 17,20-lyase conversion to dehydroepiandrosterone was observed, a temporal pattern of metabolism also observed upon expression in COS-1 cells (16). This same pattern was observed in the reconstitution experiments presented in Fig. 3. Consequently, the activities of bovine P45017a expressed in E. coli are indistinguishable from those expressed in monkey kidney cells or yeast. The flavoprotein NADPHcytochrome P450 reductase is a ubiquitous enzyme in eukaryotic cells that is capable of supporting the activity of all known microsomal forms of cytochrome P450. Furthermore NADPH-cytochrome P450 reductase from one species is able to support the activity of cytochromes P450 from other species. However, the presence of this enzyme in E. coli has not been detected immunologically (28). A unique form of cytochrome P450 in Bacillus megaterium (P450BM3) is found to be a fusion protein between a cytochrome P450 and a flavoprotein that resembles the eukaryotic NADPHcytochrome P450 reductase in primary sequence, by binding both FAD and FMN and by utilizing NADPH as a source of Table 1. Reconstitution of recombinant P45017a by adding rat liver P450 reductase or E. coli soluble fractions to membranes containing 0.5 nmol of P45017a
Prog
-*
17a-OH-Prog
conversion
Addition Rat liver P450 reductase 0.1 nmol 0.5 nmol 2.5 nmol
E. coli soluble fraction Strain JM109
Rate, nmol/min per nmol of P45017a
reducing equivalents (29, 30). Also, NADPH-sulfite reductase in Salmonella typhimurium and E. coli is a bacterial flavoprotein reported to have properties similar to NADPHcytochrome P450 reductase (31). However, a soluble protein
50 25
0
7aH-PreEA
DHlEA
v
I
I.
1 7a-hydroxypregnenolone 100
V-V
V 17ctOH-Pre, DHEA
v
7 75-
-E 0
50
-
0
25
-
c
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0
T
IIT
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% in 10 min
Progesterone 100
"
75 0.4-0.6 0.7 1.0
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S Pre,
Pregnenolone
100
63 70 90
0.4 36 Strain AT713 0.4 33 BSA
Expression and enzymatic activity of recombinant cytochrome P450 17a-hydroxylase in Escherichia coli (steroid hydroxylase/bacterial expression/reductase)
HENRY J. BARNES, MICHAEL P. ARLOTTO,
AND
MICHAEL R. WATERMAN*
Department of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235
Communicated by Ronald W. Estabrook, March 11, 1991 (received for review February 6, 1991)
When the cDNA encoding bovine microsomal ABSTRACT 17a-hydroxylase cytochrome P450 (P45017a) containing modifications within the first seven codons which favor expression in Escherichia coli is placed in a highly regulated tac promoter expression plasmid, as much as 16 mg of spectrally detectable P45017a per liter of culture can be synthesized and integrated into E. coli membranes. The known enzymatic activities of bovine P45017a can be reconstituted by addition of purified rat liver NADPH-cytochrome P450 reductase to isolated E. coli membrane fractions containing the recombinant P45017a enzyme. Surprisingly, it is found that E. coli contain an electrontransport system that can substitute for the mammalian microsomal NADPH-cytochrome P450 reductase in supporting both the 17a-hydroxylase and 17,20-lyase activities of P45017a. Thus, not only can E. coli express this eukaryotic membrane protein at relatively high levels, but as evidenced by metabolism of steroids added directly to the cells, the enzyme is catalytically active in vivo. These studies establish E. coli as an efficacious heterologous expression system for structurefunction analysis of the cytochrome P450 system.
Microsomal cytochromes P450 are integral membrane hemoproteins that catalyze the oxidative metabolism of a wide variety of endogenous and exogenous compounds. Deriving reducing equivalents from NADPH via a membrane-bound flavoprotein oxidoreductase (NADPH-cytochrome P450 reductase), these mixed-function oxidases activate molecular oxygen so as to insert one atom into a lipophilic substrate and the other atom into water. Recent study of the molecular aspects underlying eukaryotic cytochrome P450 structure and function has relied on the techniques of molecular biology to synthesize specific individual forms of cytochrome P450 in heterologous expression systems. Yeast (1), COS 1 (2), and eukaryotic cells infected with a viral vector (3, 4) have been used as hosts for the heterologous expression of cytochrome P450 molecules; however, each has limitations to their usefulness as systems for structure-function analysis. Although the bacterium Escherichia coli has demonstrated great usefulness in the expression of many prokaryotic and eukaryotic proteins, E. coli as an expression system for cytochrome P450 has been limited primarily to the soluble prokaryotic forms of this gene superfamily (5). We have used the cDNA encoding bovine 17a-hydroxylase cytochrome P450 (P45017a) to examine the utility of E. coli as an expression system for eukaryotic cytochromes P450 in the hopes that such a system might prove suitable for both enzymatic and structural studies. This microsomal cytochrome P450 catalyzes the regiospecific and stereospecific 17a-hydroxylation of the C21 steroids pregnenolone and progesterone in the pathway leading to the production of cortisol and the 17,20-lyase conversion of 17a-hydroxypreg-
nenolone to the C19 adrenal androgen dehydroepiandrosterone (DHEA) in the adrenal cortex of most mammalian species. P45017a also converts these 17a-hydroxylated products to the C19-androgen precursors of sex hormones via the 17,20-lyase reaction in the gonads of all species of mammals. P45017a [product (CYP17 in ref. 6) of the CYPJ7 gene (6)] is a typical representative of the large number of microsomal cytochrome P450 enzymes, and it is expected that knowledge gained in the study of bacterial expression of this specific cytochrome P450 will be generally applicable to this larger group.
MATERIALS AND METHODS Bacterial Strains and Plasmids. The E. coli strains used in this work were JM109 (7) and AT713 [A-, cysJ, argA21, lysA22, rpsL104, malAJ(AR), xyl-7, mtl-2] (8). The plasmid pCWOri+, a derivative of pHSe5 (9, 10), was used to express wild-type or modified P45017a cDNA sequences. This plasmid contains two tac promoter cassettes (Pharmacia no. 27-4883-01) upstream of an Nde I (CATATG) restriction enzyme cloning site coincident with the initiation ATG codon. The vector also contains a strong trpA transcription terminator sequence, a phage M13 origin of DNA replication, and the lacIq gene encoding the Lac repressor molecule that prevents transcription from the tac promoters prior to addition of inducing agents. Native and modified cDNA sequences were introduced into the expression plasmid pCWOri+ via polymerase chain reaction (PCR) mutagenesis (11). Synthetic oligonucleotides containing the native or mutant 5' cDNA sequences were used in conjunction with a downstream oligonucleotide to amplify the sequences between the ATG initiator codon (contained within an Nde I site) and a unique EcoRI restriction site of the plasmid pCD17a-2 (12). Following subcloning and sequencing of the amplification products, the expression plasmids were constructed by the simultaneous ligation of Nde I/HindIIIcleaved vector DNA with a 1257-base-pair (bp) EcoRI/ HindIll DNA fragment containing the cDNA encoding P45017a amino acids 92-509 and a 272-bp PCR Nde I/EcoRI fragment containing the native or modified cDNA PCR fragments encoding P45017a amino acids 1-91. The final expression plasmids (pCWnatl7 and pCWmodl7) were subjected to diagnostic restriction enzyme analysis prior to transformation into E. coli. Bacterial Expression and Cellular Fractionation. Ampicillin-resistant colonies of JM109 cells transformed with plasmid DNA were streaked on a fresh Luria-Bertani (LB) agar/ampicillin plate (10 g of tryptone, 5 g of yeast extract, 5 g of NaCl, 100 mg of ampicillin, and 15 g of agar per liter) Abbreviations: PCR, polymerase chain reaction; IPTG, isopropyl
/-D-thiogalactopyranoside; P45017a, 17a-hydroxylase cytochrome
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
P450 (also called CYP17), the product of the CYP17 gene; DHEA, dehydroepiandrosterone. *To whom reprint requests should be addressed. 5597
5598
Biochemistry: Barnes et al.
and were grown overnight at 370C. A single isolated colony was placed into LB/ampicillin medium and grown at 370C with shaking to near saturation. Sterile glycerol was added to this culture to a final concentration of 15% and aliquots were stored frozen at -70TC. These frozen samples served as innoculum for further expression experiments. Briefly, E. coli harboring the P45017a expression plasmids were grown to an OD550 = 0.4-0.8 in TB broth (13) containing 50-100 pug of ampicillin per ml at 370C. Where indicated, induction of the tac promoters was initiated by 1 mM isopropyl 8-Dthiogalactopyranoside (IPTG). The cells were shifted to 280C and gently shaken for 48 hr, pelleted, washed once in 50 mM 3-(N-morpholino)propanesulfonic acid (Mops) buffer (pH 7.5) containing 100 mM KCI, 1 mM EDTA, and 1 mM dithiothreitol and were resuspended in the same buffer (1/ 20th of the original culture volume). Lysozyme was added to a final concentration of 0.2 mg/ml, and the cells were incubated on ice for 30 min. Phenylmethylsulfonyl fluoride (100 mM stock in isopropanol), leupeptin, and aprotinin were added to final concentrations of 1 mM, 0.1 tug/ml, and 0.04 units/ml, respectively. The resulting spheroplasts were transferred to (2 x 4) cm glass vials placed in salt ice (-50C) and lysed by three 20-sec pulses from a 1.2-cm diameter probe attached to an Artek Sonic Dismembrator (model 150; Artek Systems, Farmington, NY) at 60% ofmaximum power. Unbroken cells and debris were pelleted at 1200 x g for 10 min. MgCl2 (6 mM) was added to the supernatant, which was centrifuged at 225,000 x g for 30 min at 4°C (14). The resultant membrane pellet was resuspended in Mops buffer containing 6 mM MgCl2 by gentle homogenization and was recentrifuged at 225,000 x g as before. After centrifugation, this washed membrane pellet was resuspended in Mops buffer. For electrophoretic analysis, a total cell protein fraction was prepared by boiling cells in 62.5 mM Tris HCl (pH 6.8) containing 2% SDS. Samples were fractionated on SDS/8% polyacrylamide gels and transferred to nitrocellulose membranes for immunoblot analysis (15). Spectral Studies. To obtain a P450 difference spectrum (23) in whole E. coli cells, cells treated with the reducing agent sodium dithonite and bubbled with CO were contained in the sample cuvette and cells treated with reducing agent alone were contained in the reference cuvette (reduced CO/reduced difference spectrum). A 100-ml culture of strain JM109 harboring pCWmodl7 was grown and induced as described above. Cells were washed and resuspended in 5 ml of Mops buffer. A 0.5-ml aliquot of concentrated cells was diluted with 5.5 ml of Mops buffer containing 10 mM glucose and was divided equally between two cuvettes. Several grains of sodium dithionite were added to each cuvette, and the baseline reduced spectrum was recorded in an Aminco DW-2A spectrophotometer. CO was then bubbled through the sample cuvette, and the reduced CO/reduced difference spectrum was recorded. For substrate binding spectra, a volume of washed cells containing 1.6 nmol of P45017a was diluted to 6 ml with Mops buffer and divided into the sample and reference cuvettes. A baseline tracing was recorded from 350 to 510 nm. Steroid was added in 10-,l aliquots (1-2 mg/ml in ethanol) to the sample with an equal volume of ethanol added to the reference. Additional aliquots of steroids and ethanol were added until the bacterially expressed P45017a was saturated as determined spectrally. Steroid Metabolism of the Recombinant Enzyme. For in vivo metabolism, E. coli were cultured and induced as described above. Cells were pelleted, washed once in Mops buffer, and resuspended in the same buffer at 1/20th of the volume of the original bacterial culture. A volume of concentrated cells corresponding to 3.7-4.2 nmol of P45017a was diluted to S ml in Mops buffer containing 10 mM glucose, 2.5 AM steroid, and 100,000 cpm of 3H-labeled radioactive steroid per ml. Samples were incubated at 280C with gentle shaking. Aliquots
Proc. Natl. Acad. Sci. USA 88 (1991)
(0.5 ml) were removed at 0, 0.5, 1, 2, 4, 6, and 8 hr after steroid addition. After extraction of cells and culture media, steroids were analyzed by HPLC as described (16). For in vitro reconstitution of enzyme activity, E. coli membrane fractions were prepared as described above (14) with the following modifications. Potassium glutamate buffer [KGB; 25 mM Tris acetate, pH 7.6/10 mM Mg(OAc)2/100 mM L-glutamic acid, monopotassium salt/0.1 mM dithiothreitol] (17) containing 10% (wt/vol) glycerol replaced Mops buffer for washing and sonication of cells. Washed cell pellets were resuspended in this buffer at a ratio of 2 ml ofbuffer per g (wet weight) of cells. A soluble protein fraction (2.9 ml/g of cells) was obtained by treating cells with lysozyme and sonication, followed by low-speed removal of debris and high-speed pelleting of E. coli membranes. This soluble fraction was dialyzed overnight at 40C against three changes of 300 ml of KGB/glycerol buffer. The dialyzed protein solution was centrifuged at 225,000 x g for 30 min to remove aggregated material, and the supernatant (now 2.4 ml/g of cells) was used as the cytosol fraction. The membrane pellet was resuspended in 0.25 M sucrose. Both cytosol and membrane fractions were stored at -70'C until use. For reconstitution of P45017a activities, membranes containing 0.5 nmol P45017a were combined with different amounts of purified rat liver microsomal NADPH-cytochrome P450 reductase (18) or with dialyzed E. coli cytosol at 370C. The P45017a/ cytosol ratio was maintained at 1:1 based on the cell mass used for initial fractionation. After a 10-min preincubation, reactions were initiated by addition of NADPH (1 mM) and steroids (5 AM plus 105 cpm of 3H-labeled radioactive steroid per ml) in a final volume of one ml of KGB or 50 mM potassium phosphate buffer (pH 7.5), each containing 12% glycerol. Samples were shaken vigorously at 370C, 200-gl aliquots were removed at various times after NADPH/ steroid addition and extracted immediately with methylene chloride, and steroids were analyzed by HPLC.
RESULTS AND DISCUSSION For expression in bacteria, the cDNA for the coding region of bovine P45017a (12) was cloned into the E. coli expression vector pCWOri+. Upon transformation of this expression plasmid containing the native codons of P45017a into the E. coli strain JM109, no immunoreactive P45017a protein was produced following derepression of the tac promoters (Fig. 1B, lane 2). Examination of the amino-terminal coding sequence of P45017a, based on the reports by others that this region plays an important role on expression levels in E. coli, led to alterations within the first seven codons of the cDNA by PCR mutagenesis as indicated in Fig. 1A in an attempt to optimize parameters for bacterial expression. Specifically, the native second codon was changed from TGG (Trp) to GCT (Ala), a preferred second codon for expression of the lacZ gene (19), and codons 4 and 5 were changed to TTA (silent mutations), since this region of E. coli mRNAs has been shown to be rich in adenosine and uridine nucleotides (20). Also the last nucleotide of codons 6 and 7 was changed to adenosine and thymidine (silent mutations), respectively, to minimize secondary structure formation in the messenger RNA (21). Immunoblot analysis (Fig. 1B) indicates that these alterations have a profound effect on the expression of P45017a in E. coli and that this expression is efficiently repressed in the absence of IPTG. Fractionation of transformed E. coli into membranes and cytosol (14) established that the expressed P45017a is associated with the membranes. Furthermore, the P45017a enzyme present in these membranes is not rendered soluble by treatment with 0.1 M sodium carbonate (pH 11.5), indicating that it is an integral membrane protein (22) in E. coli (data not shown). Only very
Biochemistry: Barnes et al.
Proc. Natl. Acad. Sci. USA 88 (1991)
teristic 450-nm absorbance maximum of all cytochromes P450 (23). Binding of substrates was also observed by detection of substrate-induced difference spectra (24) in intact bacteria after addition of the C21 steroids pregnenolone, progesterone, 17a-hydroxypregnenolone, or 17a-hydroxyprogesterone (Fig. 2B). The C19 steroid product of the bovine 17,20-lyase reaction, DHEA (16), showed much less binding, whereas another C19 steroid, androstenedione, which is not a product of bovine 17,20-lyase (16), showed no binding. The functional role of the binding of C21 steroids to the expressed P45017a was determined by incubation of transformed E. coli membranes with purified rat liver NADPH-cytochrome P450 reductase and radiolabeled substrates in a reconstitution assay followed by HPLC analysis of substrates and products. The 17a-hydroxylase and 17,20lyase activities known to be associated with bovine P45017a could be observed in reconstituted systems (Fig. 3). The turnover number of about 1.0 min-' for conversion of progesterone to 17a-hydroxyprogesterone by bovine P45017a in E. coli membranes (Table 1) is in the same range but smaller than that of 6.9 min- reported for purified pig P45017a (25). The moderate difference in turnover numbers may be due to intrinsic differences between species and/or failure to achieve maximal reconstitution of activity in bacterial membranes. Surprisingly, E. coli are capable of supporting the enzymatic activities of P45017a. The enzymatic profile (Fig. 4) of the expressed bovine P45017a in bacteria was the same as that observed in other heterologous expression systems, COS-1 cells (16) and yeast (27). Briefly, pregnenolone and progesterone were readily converted to their 17a-hydroxylated products, and 17a-hydroxypregnenolone was converted to the C19 steroid DHEA, whereas 17a-hydroxyprogesterone was not converted to androstenedione. Furthermore, when pregnenolone was added as substrate it first was converted almost entirely to 17a-hydroxypregnenolone be-
A nat17
ATG TGG CTG CTC CTG GCT GTC TTT Met Trp Leu Leu Leu Ala Val Phe
modl 7
ATG GCT CTG TTA TTA GCA GTT TTT Met Ala Leu Leu Leu Ala Val Phe
B
1
2 3 4 5 6 7 ...:..
...
17aH, l~~~~aOH'~ ~ ,,,
;
^
FIG. 1. Nucleotide and amino acid sequences at the 5'-ends of native (natl7) and modified (modl7) P45017a cDNAs and their expression in E. coli. (A) The nucleotide changes (indicated in boldface letters) introduced via PCR mutagenesis. (B) Immunoblot analysis of bacterially expressed 17a hydroxylase (17aOH). Lanes: 1, 9.4 Mg of bovine adrenocortical microsomes; 2, 50Mug of total cell protein (TCP) of strain JM109 (pCWnatl7) + IPTG; 3, 50 tg of TCP JM109 (pCWmodl7) - IPTG; 4, 50 Mg of TCP JM109 (pCWmodl7) + IPTG; 5, a mixture of samples shown in lanes 6 and 7; 6 and 7, the 225,000 x g supernatant and pellet fractions, respectively, from a 200-Ml culture of JM109 (pCWmodl7) + IPTOG.
minor proteolytic degradation of P45017a is evidenced by immunoblot analysis. Not only is the P45017a protein expressed in E. coli, but also this protein has the spectral characteristics of functional P45017a (Fig. 2j. The reduced CO/reduced difference spectrum obtained in intact E. coli (Fig. 2A) shows the charac-
A
5599
B Preg 17caOH-Preg
1~~~~~~~~~~0.01
0
DHEA
0.01
Baseline
370
a -0
390
410
430
450
Baseline
co Qa
Co
0
-0
E1 7aOH-Prog
.0~~~~~~~~~~00
0
AD _____________________________________
420
450
Wavelength (nm)
480
370
__
Baseline
390 410 430 450 Wavelength (nm)
FIG. 2. Reduced CO/reduced difference spectrum (A) and substrate binding spectra (B) of P45017a in intact E. coli cells. Steroids and their final concentrations in B are pregnenolone (Preg), 22MLM; 17a-hydroxypregnenolone (17&OH-preg), 40MAM; DHEA, 46MLM; progesterone (Prog), 21 MM; 17a-hydroxyprogesterone (17aOH-prog), 40 MM; and androstenedione (AD), 46 uM.
5600
2\/~
Proc. Natl. Acad. Sci. USA 88 (1991)
Biochemistry: Barnes et al.
4--
0 0
._
0o
0
30
1 5
45
60
2
0
Incubation, min
4
6
8
1 0
Incubation, min
FIG. 3. Steroid metabolism of isolated E. coli membranes supplemented with purified rat liver NADPH-cytochrome P450 reductase. Reactions were performed in KGB/glycerol buffer at a reductase/P45017a molar ratio of 1:1. Initial substrate concentrations were 5 ,uM.
fore 17,20-lyase conversion to dehydroepiandrosterone was observed, a temporal pattern of metabolism also observed upon expression in COS-1 cells (16). This same pattern was observed in the reconstitution experiments presented in Fig. 3. Consequently, the activities of bovine P45017a expressed in E. coli are indistinguishable from those expressed in monkey kidney cells or yeast. The flavoprotein NADPHcytochrome P450 reductase is a ubiquitous enzyme in eukaryotic cells that is capable of supporting the activity of all known microsomal forms of cytochrome P450. Furthermore NADPH-cytochrome P450 reductase from one species is able to support the activity of cytochromes P450 from other species. However, the presence of this enzyme in E. coli has not been detected immunologically (28). A unique form of cytochrome P450 in Bacillus megaterium (P450BM3) is found to be a fusion protein between a cytochrome P450 and a flavoprotein that resembles the eukaryotic NADPHcytochrome P450 reductase in primary sequence, by binding both FAD and FMN and by utilizing NADPH as a source of Table 1. Reconstitution of recombinant P45017a by adding rat liver P450 reductase or E. coli soluble fractions to membranes containing 0.5 nmol of P45017a
Prog
-*
17a-OH-Prog
conversion
Addition Rat liver P450 reductase 0.1 nmol 0.5 nmol 2.5 nmol
E. coli soluble fraction Strain JM109
Rate, nmol/min per nmol of P45017a
reducing equivalents (29, 30). Also, NADPH-sulfite reductase in Salmonella typhimurium and E. coli is a bacterial flavoprotein reported to have properties similar to NADPHcytochrome P450 reductase (31). However, a soluble protein
50 25
0
7aH-PreEA
DHlEA
v
I
I.
1 7a-hydroxypregnenolone 100
V-V
V 17ctOH-Pre, DHEA
v
7 75-
-E 0
50
-
0
25
-
c
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0
T
IIT
a)
0m
% in 10 min
Progesterone 100
"
75 0.4-0.6 0.7 1.0
V 1
JV
75
0_0
S Pre,
Pregnenolone
100
63 70 90
0.4 36 Strain AT713 0.4 33 BSA
