Expression and Purification of Functional Human 17a=Hydroxylase ...

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Sep 15, 2017 - Reproductive Biology Sciences, University of Texas Southwestern Medical Center, Dallas, ... 5 Present address: Department of Surgery 11, Nagoya University ...... Poulos, T. L., Finzel, B. C., and Howard, A. J. (1987) J. Mol. ... Youngblood, G. L., Sartorius, C., Taylor, B. A., and Payne, A. H. (1991) Genomics.
Vol. 268, No. 2 6 , Issue of September 15, pp. 19681-19689, 1993 Printed in U.S.A.

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc

Expression and Purification of Functional Human 17a=Hydroxylase/ 17,20-Lyase (P450c17)in Escherichia coli USE OF THIS SYSTEM FOR STUDY OF A NOVEL FORM OF COMBINED 17a-HYDROXYLASE/17,2O-LYASE DEFICIENCY* (Received for publication, February 3, 1993)

Tsuneo ImaiS§, Hadas Globermannll, Joseph M. Gertned, Norio KagawaS**, and Michael R. WatermanS SS From the $Departments of Biochemistry and Obstetrics and Gynecology and The Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, Dallas, Texas 75235 and the llDepartment of Pediatrics, Pediatric Clinical Research Center, New York Hospital-Cornell Medical Center, New York. New York 10021

onchromosome10(Matteson et al., 1986).Thefull-length 1527-bp P450c17 cDNA encodes a protein of 508 amino acids (Bradshaw et al., 1987). P450c17 has been purified from pig adrenal (Nakajin et al., 1984), pig testes (Nakajin et al., 1981; Katagiri et al., 1982; Suhara et al., 1984), guinea pig adrenal et al., 19911, a n d (Kominamiet al., 1982), and calf testes (Perrin has been shown to be a single polypeptide which can catalyze both l7a-hydroxylation of pregnenolone and progesterone and the subsequent 17,20-lyase reactions converting the resultant 17a-hydroxysteroids to the C19 androgens, dehydroepiandrosterone(DHEA),andandrostenedione.Thesedualactivities have been confirmed by transfecting and expressing P450c17 et al., 1986a), yeast (Sakaki et al., 1989), cDNAin COS-1 (Zuber or Escherichia coli (Barnes et al., 1991). Human, bovine, and pig forms of P450c17 contain more than 75% homology in primary amino acid sequence; however, certain differences in enhas zymatic properties exist within those species. Pig P450c17 17,20-lyase activity for both A 5 (17a-hydroxypregnenoloneto DHEA) andA4 (17a-hydroxyprogesteroneto androstenedione) pathways, as do t h e rat (Fevold et al., 1989) and trout (Sakai et al., 1992) enzymes. On the other hand, bovine and human the A4 pathway P450c17 have little 17,20-lyase activity for upon expression COS 1 cells (Fevold et al., 1989; Yanaseet al., 1991) andE. coli (Barnes et al., 19911, as has also been shown for the purified enzyme from calf testes (Perrinet al., 1991). One approach to the study of structure/function relationships of P450c17, is to couple site-directed mutagenesis with heterologous expression (Yanaseet al., 1989; Clark and Waterman, 1991; Kitamura et al., 1991). However, many expression systems produce relatively small amountsof P450c17, making Humansteroid 17a-hydroxylase/l7,2O-lyase(P45Oc17)’ is spectrophotometric analysisof m u t a n t s difficult. In an effort to of P450c17 for detailed structure/ encoded by a single gene, CYP17 (Nebert et al., 19911, located generate sufficient quantities functionanalyses,wehavedeveloped a n E. coli expression * This work was supported in part by Research Grant 1-FY92-0907 system (Barneset al., 1991). We have now applied this system from the March of Dimes Birth Defects Foundation and United States to expression of h u m a n P450c17 and have establishedan effiPublic Health Service Grants GM37942 and RR06020. The costs of of histidine publication of this article were defrayed in part by the payment of page cient affinity purification procedure by addition charges. This article must therefore be hereby marked “aduertisernent” residues at the carboxyl terminus for this previously inaccesin accordance with 18 U.S.C. Section 1734 solely toindicate this fact. sible form of P450c17. For human P450c17, an additional ap5 Present address: Department of Surgery 11, Nagoya University proach to investigation of structure/function relationships is School of Medicine, Nagoya 866,Japan. /I Present address: Department of Pediatrics, Carmel Hospital, Haifa, the characterizationof naturally occurring mutations (molecular basis of 17a-hydroxylase deficiency) followed by heteroloIsrael. ** Present address: Dept. of Biochemistry, School of Medicine, gous expression studies (Yanase et al., 1989, 1992; Ahlgren et Vanderbilt University, Nashville, TN 37232. al., 1992;Lin et al., 1991).SinceBiglieri et al. (1966) first $$ To whom correspondence should be addressed. Present address: Dept. of Biochemistry, School of Medicine, Vanderbilt University, 607 Light Hall, Nashville, TN 37232-01467. Tel.: 615-322-3318; Fax: 615topyranoside; 170H-preg, 1701-hydroxypregnenolone;170H-prog, 17a322-4349. hydroxyprogesterone; DHEA, dehydroepiandrosterone; ALA, 8-aminThe abbreviations used are: P450c17, 17a-hydroxylasecytochrome olevulinic acid; NTA, nitrilotriacetate; PAGE, polyacrylamidegel P450; PCR, polymerase chain reaction; IPTG, isopropyl P-D-thiogalac- electrophoresis; bp, base pair(s).

Enzymatically activehumanl7a-hydroxylase cytochrome P450 (P450c17)has been expressed in and purified from Escherichia coli. The cDNA containing modifications withinthe amino-terminal eightcodons which are favorable for expressionin E. coli, as well as codons for 4 histidine residues at the carboxyl terminus, was placed in thepCWori+ expression vector. The modified human P450c17 was detectedspectrophotometrically (400 nmol of P450c17fliter culture) and was found to be integrated intoE. coli membranes. This previously inaccessible human P450 was purified to electrophoretichomogeneity (10.7 nmol of P450/mg) from solubilized bacterial membranes using two sequential chromatographic steps, nickel nitrilotriacetate followed by hydroxylapatite. The expected enzymatic activities of human P450c17 were reconstitutedby addition of purified rat liver NADPH-cytochromeP450 reductase, giving turnover numbers of 8.0 nmol/min/nmol P450 for pregnenolone, 6.5 nmol/min/nmol P450for progesterone,0.06 nmol/min/nmol P450 for 17a-hydroxypregnenolone,and nodetectableactivityfor 17a-hydroxyprogesterone. This system was utilized to study the molecular basis of a novel form of combined 17a-hydroxylase, 17,20-lyase deficiency resulting from compound heterozygous mutations, a missense point mutation Tyre4(TAT)+ Ser (TCT),and anIle112duplication (ATCATC).Upon expression of these mutant proteins E. incoli, the Tyr64mutant has 15%of the wild type 17a-hydroxylase activity, whereas the Ile112 duplication shows no activity, results consistent with the observed clinical phenotype.

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Expression and Purification of Human P450c17 in E. coli

19682

reported l7a-hydroxylase deficiency, morethan 150 cases have been discovered (Yanase et al., 1991). To carefully study the enzymatic properties of either naturally occurring mutants or those generated by site-directed mutagenesis, purification of the enzyme is essential. Here we report the heterologous expression of functional human P450c17 in E. coli upon modification of its NH2 terminus, and its purification, by a simple chromatography system including metal-immobilized affinity chromatography by addition of 4 histidine residues to the COOH terminus. We also report elucidation of previously undescribed compound heterozygous mutations as the cause of complete combined17a-hydroxylase/l7,20-lyasedeficiency and the use of the expression and purification procedures to study the enzymatic properties of mutant forms of P450c17. EXPERIMENTALPROCEDURES Materials-Restriction endonucleases and other modifymg enzymes were purchased from Boehringer Mannheim. Taq polymerase was obtained from Perkin-Elmer Cetus. [3HlPregnenolone,[3Hlprogesterone, [3H117a-hydr~~ypregnen~lone, and PHI 17a-hydroxyprogesteronewere purchased from ICN Biochemicals (Costa Mesa, CA). Prestained protein molecular weightstandards was purchased from Life Technologies, Inc. Construction of Recombinant Expression Plasmids in E. coli-The plasmid pCWOri+, a derivative of pHSe5 (Muchmoreet al., 1989),was used to express modified human P450c17 cDNA sequences (Barnes et al., 1991). ModifiedcDNA sequences were generated via polymerase chain reaction (PCR) mutagenesis (Higuchi et al., 1988). Synthetic oligonucleotides containing the modified 5' cDNA sequences (Fig. IA) were used in conjunction with a 3'-oligonucleotide to amplify the sequences between the ATG initiation codon (contained within an NdeI site) and a unique XbaI restriction site in the human P450c17 cDNA (Bradshaw et al., 1987), using pUCHl7a-1 (Yanase et al., 1989) as a template. The PCR products were subcloned into the HincII site of pBluescript SK(-)(Stratagene, San Diego, CA) and the amplified sequences confirmed by dideoxynucleotide sequence analysis. A 306-bp partially digested NdeIIXbaI fragment (there is another NdeI site in this sequence)encodinghuman P450c17 amino acids 1-102, was ligated into pCWOri+. Then a 1420-bpXbaIIHindIII fragment of pUCHl7a-1, encoding human P450c17 amino acids 103-508, was subcloned to construct plasmid pCWHl7mod. Additionalsynthetic oligonucleotides, one encoding 4 histidine residues plus the stop codon (overlappingthe HindIII site) andone 568 bpupstream, were used to amplifyand construct the COOH-terminal modification (Fig. 1 B ) . Aunique SacIIHindIII fragment (193 bp) fromthe PCR fragment was subcloned into pCWHl7mod, producing pCWHli'mod(His),, whichwas subsequently sequenced. Expression ofpCWH17modin E. coliand Membrane Isolation-Each experiment was begun by picking a single colony of bacteria from a Luna-Bertani (LB) plate (Sambrook et al., 1989) containing 50 pg/ml ampicillin and seeding into LB broth. Overnight cultures (U100 volume) at 37 "C were seeded into terrific broth (TB) (Tartof and Hobbs, 1987)containing 50 pg/ml ampicillin. Bacteria were growna t 37 "C for 2.5 h using a shaking rate of 240 rpm (A,,, reached between 0.4 and 0.8). Isopropyl-P-o-thiogalactopyranoside(IPTG) and Saminolevulinic acid (ALA) were added to 1m~ to induce the expression of P450c17. The 1 A

lhlpan

native 17

A M TQQ QAQ CTC O M

Yet T ~ R Qlu

mu

QCT CTC TTQ

va1 Ala ~ e uLOU

modified I

AMGCTWZTAZTAQCAWFTTI! Met U &u m u Ala ynl m

modified I1

AM

pcIIAl7rod

ACC T m

GSZ W ZTA =A Oca C l Z TT(I Hat U Qlu Leu Lw Ala Leu Leu

508 B

Thr etop

,

....................... Hind111 I

206bp untranslated eewence 508

pcIIAl7pod(IiiSh

512

ACC CAT CAC CAT CAC

Thr

*top Hind111

FIG.1. Modifications of human P4M)Cll at theN H a and COOH termini. Nucleotide and predicted amino acid sequences at the 5' end (A) and the 3' end ( E )of human P450c17 cDNAsare indicated with the nucleotide changes introduced via PCR mutagenesis being underlined.

temperature was then shifted to 30 "C and shaking rate decreased to 120 rpm. After 24 h of incubation, bacteria were chilled and harvested by centrifugation. For measurement of reduced CO difference spectra and Western analysis, bacteria were resuspended in anequal volume of 0.1 M sodium phosphate, pH 7.2, 20% glycerol. For isolation of membrane fractions, bacterial pellets from100-ml cultures were resuspended with 10 ml of TES buffer (0.1 M Tris-acetate, pH 7.8, 0.5 mM EDTA, 0.5 M sucrose). Lysozyme was added to 0.2 mg/ml with 10 ml of ice-cold water followed by incubation for 30 minon ice with continuous mixing. Spheroplasts were harvested by centrifugation (12,000 x g for 10 mid, and resuspended in 4ml of ice-cold 0.1 M sodium phosphate, pH 7.2, containing 20% glycerol,0.5 mM phenylmethylsulfonylfluoride, and 10 pg/ml aprotinin. After freezing and thawing, samples were sonicated on ice (pulse 20 s on, 30s off, repeated 6 times), using a sonicator with a 1.2-cm titanium probe (Artek Sonic Dismembrator, model 150,Artek Systems, Farmington, NY) at 70%of maximum power. Unbroken cells and debris were pelleted at 3000 x g for 10 min, and the supernatant was centrifuged at 225,000 x g for 30 min at 4 "C (Beckman TL-100). The pellets were washed with the same buffer and resuspended with 1 ml of 0.1 M sodium phosphate, pH 7.2,20% glycerol using a Teflon-glass homogenizer. Purification of P450H17mod(His)-Five ml overnight 37 "C culture was seeded into 500 ml of TB medium with 50 pg/ml ampicillin in a Fernbach 3.8-liter flask. Bacteria were grown as above, and 24 h after IPTG and ALA addition, bacteria from 1 liter of culture were chilled, harvested by centrifugation, and resuspended in 100 ml of ice-cold TES buffer. After lysozymetreatment, spheroplasts were harvested and resuspended using a glass-glass homogenizer on ice in 25 ml of ice-cold buffer A (0.1 M sodium phosphate, pH 7.2, 0.1 M NaCl, 20% glycerol,50 p~ progesterone) with 1%Emulgen 913 (Go-Atlas, Tokyo, Japan). At this stage, samples would be kept at -70 "C until further use (volume was about 33 ml from 1liter of culture). Frozen samples from 9 litersof culture were thawed, and aprotinin and phenylmethylsulfonyl fluoride were added to 0.1 mg/ml and 0.5 m ~ respectively. , Spheroplasts were sonicated on ice with stirring (pulse 1 min on, 2 min off, repeated 8 times), and then centrifuged at 193,000 x g for 1h at 4 "C (Beckman Ti 50.2 rotor). The supernatant was passed through a 5-pm filter and diluted with an equal volume of buffer A without Emulgen 913 (final concentration of Emulgen 913 was 0.5%), and applied to a nickel nitrilotriacetate (Ni2+-NTA)agarose (Qiagen Inc., Chatsworth, CA) column (2.2 cm in diameter, bed volume 35 ml). The column was washed with 10 volumes of buffer A containing 50 mM glycine, 0.2%Emulgen 913, and eluted with buffer Acontaining 40 mM histidine, 0.2%Emulgen 913. The fractions having an A,,, over 0.3 were collected,diluted with four volumes ofH,O containing 20% glyceroland 50 PM progesterone, and immediately applied to a Bio-Gel HTP hydroxylapatite (Bio-Rad) column (2.6 cm in diameter, bed volume 30ml). The column waswashed with five volumes of buffer B (80 mM sodium phosphate, pH 7.2, 20% glycerol, 0.02% Emulgen 913, 50 p~ progesterone), and eluted slowly with buffer B containing 300 mM sodium phosphate. The concentrated fractions were collected and dialyzed overnight a t 4 "C against 4 liters of buffer B containing 0.1 M sodium phosphate with one bufferchange. Emulgen 913 and progesterone were removed by hydroxylapatite chromatography before enzyme reconstitution assay and measurement of substrate binding difference spectra. P450H17modVBUHi~)~ was purified from a 1.5-liter culture using the same protocol. ReconstitutionAssay of Human 17a-Hydroxylasel17,20-Lyase Activities-17a-Hydroxylase activity was analyzed as follows. 50 nM P450 and 150 nM rat liver P450 reductase (kindly provided byDr.R. W. Estabrook) were mixed and preincubated in 20 pl of R buffer (50 mM sodium phosphate, pH7.2,20% glycerol, 10 mM magnesium acetate, 100 pg/ml L-a-lecith'n phosphatidylcholine) at 37 "C for 2 min. Substrate was mixedwith 3H-labeledsubstrate (50,000 cpm) in Rbuffer, added to the reaction mixture (200 pl final volume), and preincubated at 37 "C for 5 min before the reaction was started by addition of NADPH (final 1mM). The reaction was carried out a t 37 "C for10 min and terminated by adding cold methylene chloride. 17,20-Lyase activity was analyzed as described above, except 50 to -200 nM final concentration of P450 and a 3M excess of P450 reductase were used for the reaction a t 37 "C for 30 min. Steroids were extracted in 10 volumes of dichloromethane with excess cold A4 (progesterone, 170H-prog, androstenedione) or A5 (pregnenolone, 170H-preg, DHEA) mixtures and evaporated to dryness. Steroids were dissolved with 100 pl of acetone and separated on thin layer chromatography (TLC) (EM Science, Silica Gel 601-254for A4 products, and Whatmann Silica Gel 60A for A5 products). The solvent systems were ch1oroform:ethylacetate (80:20) forA4 steroids and (955) for A5 steroids (McCarthy and Waterman, 1988). The location of separated steroids were determined by U V light (A4), or iodine vapor (A5). TLC

Expression and Purification

of Human P450c17 E.in

strips were cut out and immersed in 0.5 ml of methanol and counted with 10ml of Budgetsolve (RPI Corp., Mount Prospect, IL) using a liquid scintillation counter. Patient VB-The Caucasian patient (VB)was recognized as female at birth. At seven weeks, repair of bilateral inguinal hernia was done and during the operation, gonads were found, which upon biopsy revealed normal testicular tissue. At 10 months the patient was admitted for evaluation at the New York Hospital-Cornell Medical Center and the karyotype was determined to be 46 XY. On physical examination, the external genitalia were normal female. By pelvic ultrasound, a uterus was not identified. Serum and urinary steroid measurements are summarized in Table I. Androgens, 17-hydroxysteroids,and cortisol were at very low basal levels, and, after stimulation by human chorionic gonadotropin or adrenocorticotropin, those steroids were increased only a little. On the other hand, desoxycorticosterone and corticosteronewere very high. Basal adrenocorticotropinwas high, and basal renin activity was low. All these data suggested that this patient was suffering from 17a-hydroxylase deficiency. Consequently, it was advised that the patient be raised as female and that thegonads be removed. At the age of 2, gonadectomywas performed, and resected testes was kept at -70 "C for further study. Preparation of DNA, RNA, and the Postnuclear Pellet Fraction from VB %tis-The weight of one frozen testis was approximately 500 mg. RNA was extracted from 200 mg by an acid guanidinium thiocyanatephenol-chloroform extraction method (Chomczynskiand Sacchi, 1987). Another 200 mg was homogenized with 0.25 M sucrose and separated into nuclear and postnuclear fractions. Genomic DNA was extracted from the nuclear fraction by treating with proteinase K, RNase A, and SDS (Sambrook et al., 1989).The postnuclear fraction was centrifuged (225,000 x g), and the pellet was resuspended with 0.25 M sucrose and used for Western analysis. (5 pg) Southern, Northern, and WesternAnalyses-GenomicDNA was digested with EcoRI and electrophoresedin a0.7%agarose gel and transferred to nitrocellulose filter. Total testis RNA (10 pg) was denatured with glyoxal, electrophoresedin a 1%agarose gel, and transferred to nitrocellulose filter. Each filter was hybridized with full-length human P450c17 cDNA,which was labeled with [a-32PldCTPusing random hexamer and Klenow enzyme (Feinberg and Vogelstein, 1983).Hybridization was carried out with 50% formamide and 5 x Denhardt's solution at 42 "C overnight. Filters were washed at high stringency and then were exposed to Kodak X-AR film with an intensifying screen at -70 "C. Southern analysis showed the expected 5.7- and 6.9-kilobase bands (Kagimoto et al., 1988) indicating no gross abnormalities in the VB CYP17 gene (data not shown). Northern analysis showed little expression of CYP17 transcripts in the VB testis, whereas control RNA from human fetal adrenal showed the expected 1.8-kilobasetranscript (data not shown). The postnuclear fraction (200 mg) was denatured with Laemmli buffer and electrophoresed in 10% SDS-PAGE gel and transferred to a nitrocellulose filter. After a blocking step with 5% dried milk, the filter was hybridized first with an antibody against purified P450H17mod(His), (15000dilution) and second with horseradish per-

coli

19683

oxidase conjugate-protein G (1:3000 dilution, Bio-Rad) in 50 mM TrisHCI, pH 7.6, 150 mM NaC1, 0.1% Tween 20. After washing using the Tris-NaC1-Ween 20 buffer, the filter was incubated with enhanced chemiluminescence (ECL) Western blotting detection reagents (Amersham Corp.) for 1 min and exposed to Kodak X-ARfilm for 20 s. The primary antibody was produced in a New Zealand White female rabbit using 1mg of purified protein injected with complete Freund's adjuvant followed by a booster using incomplete Freund's adjuvant (Harlow and Lane, 1988). The IgG fraction was obtained from the anti-sera using a protein A-Sepharose 4 Fast Flow Column (Pharmacia LKB Biotechnology Inc.) (Goding, 1976). The postnuclear fraction from the VB testis contained a low level of immunoreactive P450c17, which was the same size as that inhuman fetal adrenal (data not shown). Fig. 2 shows the comparison of cross-reactivity of anti-human and anti-pig P450c17 antibodies against P450c17 from different species expressed in E. coli. As can be seen, the available anti-pig antibody clearly recognizes rat and bovine P450c17 but recognizes human P450c17 only very poorly. Consequently it was necessary to prepare an antibody against human P450c17 for detailed study of this protein rather than relying on the anti-pig P450c17 provided to us byDr. J. Ian Mason, University of Texas Southwestern Medical Center, Dallas, T X . As seen in the left panel, the antibody against human P450c17 readily recognizes P450c17 from human, bovine, pig, and rat, most strongly recognizing human P450c17. cDNA Synthesis, cDNA Amplification, Cloning,and Sequencingfrom Testes RNA-cDNA synthesis from 1 pg of total RNA and subsequent PCR amplification of the cDNA was carried out using the GeneAmp RNAPCR Kit (Perkin-Elmer Cetus) according to the manufacturer's instructions. Reverse transcription, using oligo-d(T)le oligonucleotide as primer was performed by annealing at 22 "C for10 min, extension at 42"Cfor 60 min, and denaturation at 99 "C for 10 min, followed by storage on ice until PCR amplification. For PCR, two sets of oligonucleotides based on the known cDNA sequence were used as primers t o generate overlapping products that included the complete coding se50 quence of human P450c17 (Fig. 3). Amplification conditions included cycles of denaturation at 94 "C for 1 min, annealing at 55 "C for 1min, and extension at 72 "C for 1min. The PCR products were filledin with Klenow enzyme and were subcloned into SmaI-digested pUC19followed by transformation in TG1 bacteria. Six clones of the NHz-terminal fragment and five clones of the COOH-terminal fragment of the P450c17 cDNA were sequenced individually by the dideoxy chain termination method (Sanger et al., 1977). Sequenceanalysis revealed that three of the NHz-terminal clones contained an A + C transition at nucleode 191 changing tyrosine (TAT) to serine (TCT) at position 64 (Fig. 4A). The other three NH,-terminal clones contained A at 191 but a three-base duplication (ATC) at the position of codon 112 resulting in an isoleucine duplication at this site(Fig. 4B).The remainder of the six NHz-terminal sequences and all five COOH-terminal sequences were normal. Thus VB is a compound heterozygote having the Tyr- + Ser mutation on one CYP17allele and the isoleucine duplication at position 112on the other. Neither of these mutations have previouslybeen reported to be a cause of 17u-hydroxylasedeficiency (Imai et al., 1993).

TABLE I Plasma and urinary hormone determinations of VB. Basal values and results of ACTH and hCG stimulation tests The abbreviations used are: ACTH, adrenocorticotropin;hCG, human chorionic gonadotropin; 17 OH-Preg, 17a-hydroxypregnenolone;170HProg, 17a-hydroxyprogesterone;DHEA, dehydroepiandrosterone; DHEA-S, dehydroxyepiandrosterone sulfate; T,testosterone; DHT, dihydrotestosterone; DOC, deoxycorticosterone;B, corticosterone; F, cortisol; Aldo, aldosterone; PRA, plasma renin activity; 17KS, 17-ketosteroids;17OHCS, 17-hydroxycorticosteroids;-, not determined. Basal

Plasma hormones 17OH-Preg 170H-Prog DHEA DHEA-S T DHT DOC B F Aldo ACTH PRA Urinary steroid 17KS 17OHCS

0.60

0.31 3.7 561 16 655 191 0.42 4.5

5.2

hCG test

ACTH

(60 m i d

1.4

-

0.35

0.14 4.0 909 30 1198

-

-

24 h

1.5 0.33 0.24 380 0.31 0.13

-

48 h

0.78

-

0.18 490 0.24 0.1

-

Normal basal range

0.36-3.10 nmoVliter 0.12-3.45 nmoVliter 0.31-1.46 nmoVliter 27-490 nmoVliter 0.07-0.90 nmoVliter 0-0.25 nmoVliter

-

0.12-1.48 nmoVliter 14-58 nmoVliter 157-690 nmoVliter 140-3660 pmoVliter 4-22 pmoVliter 0.83-1.67 ng/(L.S)

3.8 1.4

0.3 -3.5 mmol/d 5.5 -11 mmol/d

-

Expression and Purification of Human P450cl7 in

19684

Anti Human MW P a )

E. coli

Anti Pig

MW

H B P R HH FB AP R H F A

(KDa)

117

117

75.5

75.5

48

48

2s

28

FIG.2. Immunoblot analysis of P4M)c17. Immunoreactivity using anti-human P450c17 antibody (left) and anti-pig P450c17 antibody (right) against human( H ) ,bovine (E), pig (PI, rat(R), forms of modified P450c17expressed in E. coli, and human fetaladrenal microsomes (HFA). Each E. coli sample is themembrane fraction containing 1pmol of P450. Human fetal adrenalmicrosomes contain 0.45 pg of protein. All samples were separated on the same gel, and, after transfer to the nitrocellulose, the filter was divided and probed with the respective antibodies. Tyr eSer (64)

ile duplication (1 12) AUG

0

UAA

250

500

750

1000

1250

1500 bD

N terminal fragment (1146 bp) C terminal fragment (810 bp) FIG.3. Strategy for amplification of human P45Oc17 M A . The hatched box indicates protein coding sequence. Bars labeled N terminal fragment and C terminal fiagmentshow the size and spatialdistribution of the two fragments amplified by PCR of cDNAprepared from RNA from the patient V B . The arrows show the sites where the two mutations were identified. The empty boxes at the ends of each bar represent the PCR primers.

The plasmids containing each mutation were named as pUCH17VB1 ( T y P + Ser) and pUCH17VB2 (Ile112duplication). Amplification and Direct Sequencing of VB Patient’s Genomic DNA -To confirm these mutations, genomic DNA (0.1 pg) was amplified using sets of primers for exon1and for exons 2-4 (Saiki et al., 1988).For exon 1, one was complementary to nucleotide sequences in the 5’-untranslated region and the other complementary to sequences in the 5’ end of intron 1. For exons 2-4, one primer was complementary to sequences in the 3’ end of intron 1 and the other complementary to sequences in the 5‘ end of intron 4. The second asymmetrical PCR amplification and sequencing of the PCR products were as previously described (Imai et al., 1992).As shown in theright-hand panels of Fig. 4 (A and B ) , direct sequencing of genomic DNA showed double bands of A and C a t nucleotide 191 and thedouble bands of the ATC duplication in exon 2. Construction of pCWHl7‘modvB and Enzyme AssayspCWH17modVBl was constructed in the same way as described for pCWHl7mod using pUCH17VB1 as a template of PCR instead of pUCH17a-1. pCWHl7modVB2 was prepared by in vitro mutagenesis using the method of Kunkel (1985). The BamHYHindIII fragment of pCWHl7mod, which includes the entire sequence of modified human P450c17, was subcloned to pBluescript SK(+)and transformed to CS236 bacteria followed by single-stranded DNA isolation. A synthetic antisense oligonucleotide containing the Ile112 duplication was used as a primer to amplify the mutagenic strand that was transformed into XL1-Blue bacteria. Eight clones were sequenced, and the XbaYSacI fragment of a correct clone was re-subcloned to pCWH17mod. A SacY Hind111 fragment of pCWHl7mod(His), was subcloned to pCWHl7modVBl to construct pCWHl7modVBl(His),. For measurement of enzymatic activity using whole cells, the pellet from 12 ml of culture media following 24h of incubation after IPTG and ALA addition was resuspended in 2 ml of TB medium and incubated as

described previously (Barnes et al., 1991). Other Methods-Protein concentrations were determined by the bicinchoninic acid method (Pierce Chemical Co.) with bovine serum albumin as standard.Reduced CO difference spectra, absolute absorption spectra, and substrate-induced difference spectra were carried out using a Cary model 118 spectrophotometer and measured according to Sato and Omura (1978). RESULTS

Expression ofpCWHl7mod in E. coli-Specific NH2-terminal sequences seem to be necessary to obtain high level expression of P450 in E. coli. The combination of the first 24 bp of the modified bovine P450c17sequence and the pCW vector is very favorable for expression of some P450s (Barnes et al., 1991; Fisher et al., 1992; Richardson et al., 1993). When preciselythe same modified bovinesequence was used in place of the normal human P450c17 sequence (Fig. IA), expression as high as 400 nmol of P450fliter of culture was achieved. This modification (I) results in 5 amino acid changes within the first 8 amino acids of human P450c17. We tried a more conservative modification (2 amino acid changes within first 8 amino acids, midification 11), but obtained only 1/10 the level of expression (40 nmol of P450fliter of culture, data not shown) compared with modification I. To purify the expressed P450 using immobilized metal ion chromatography, 4 histidine residues were placed atthe COOH terminus by PCR mutagenesis (Fig. 1B). P450H17mod(Hi~)~ was expressed at the same level as P450H17mod and both are integrated intothe membranes of E.

Expression and Purification of Human P450c17E. in

coli

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A Clone 1

Clone 2 Dlrect Sequence

(Mutated Allele)

(Normal Allele)

1/"

C

-7:

T A C

T C i

*A

*

Tyr-64

c J Sera4

A T C] T

T T C

T

r

T

B Direct Sequence

Clone 1 (Mutated Allele) 5'

,

1:

Lvs-110

3#

"_

IAI

"

\: 3' \:

fi

114112

c

Al8-113

T

Phe-114

\%

Phe-115

3'

FIG.4. The mutations identified in the CYPI7 gene from VB testis. Clone 1and clone 2 are from different allelesamplified and subcloned from testisRNA. A , clone 1 has normal sequencea t position 64 (*), but clone 2 has the mutatedone (*I. Direct sequence of genomic DNA clearly shows the compound sequence (+). B , in contrast, clone 2 has normal sequencea t position 112 (+), but clone 1has the three-base duplicationa t this position (+I. Direct sequence of genomic DNA shows the compound sequences (+I. TABLEI1 Purification of P450H17rnod(Hi~)~ from E. coli Table shows results of purification from 9 liters of culture medium following expression of pCWHl7mod(His), in DH5a bacteria. Preparation Protein

6.4

Whole bacteria Solubilized5000 sample 25 Ni-NTA 682 555 Hydroxylapatite 56

P450

Yleld

Specific content

nmol P45Olmgprotein

mg

nmol

%

15,300

2700 1165

100 43

0.18 0.23

106

-17

MW (kDa)

+17

After Ni

After 2x HA

240

111 75.5

20

10.7 48

coli (data not shown)as previously demonstrated withmodified bovine P450c17 (Barnes et al., 1991). Purification of P45OH17mod(His)-Table I1 summarizes the purification of P450H17mod(His), from 9 liters of culture. ARer a single pass over the Ni2+-NTAcolumn, most of the bacterial proteinswere removed (Fig. 5). Ni2+-NTA has a veryhigh affinity, and through a single column the specific content of P450C17mod (His)4 was increased from 0.23 to 6.4 nmol/mg protein. Although expressed P450H17mod(His), wasintegrated in E. coli membranes, we omitted isolation of the membrane fraction of E. coli during thepurification procedure. We also skipped dialysis between the two columns, by simply reducing the phosphatebuffer concentration by dilution prior to application to the hydroxylapatite column. The omissions of isolation of membrane fractions and dialysis made it possible to purify P450c17 from the frozen lysozyme-treated bacterial pellets within a single day (10 h). The major contaminants aRer

20

19

FIG.5. SDSPAGE of purified P450Hl7m0d(His)~. A , nontransformed (-17) and transformed (+17) DH5a bacteria (5 pl of culture medidane). AFFOUJ indicates the expressedP450H17mod(His), in lane +17. B, purified human P450H17mod(His), samples. Eluent from Ni2+NTA (After N i ) and hydroxylapatite(After2 x HA) columns. Each lane contains 1 pg of protein. Gels were stained with silver nitrate.

19686

Expression and Purification of Human P450c17 in E. coli

Ni2+-NTA chromatography were larger proteins, which were separated from the P450 on the hydroxylapatitecolumn. Purified P450H17m0d(His)~ shows a molecular weight of 54,000 on SDS-PAGE, and has a,specific contentof 10.7 nmol of P45O/mg of protein. Purified, oxidized P450H17mod(Hi~)~ is a mixed spin typeP450 having a Soret absorbance maximum at 410 nm in the presence of 50 PM progesterone (Fig. 6). The dithionitereduced form also has a maximum at 410 nm. TheCO complex of the dithionite-reduced form has a maximum at 448 nm with a shoulder but no peak at 420 nm. Binding of substrates was observed as classical Type I substrate-induced difference spectra (Narasimhulu etal., 1965) after additionof either pregnenolone, 170H-preg, progesterone, or170H-prog.Thesesubstrate-induced difference spectra were used for the estimation of K, values for each substrate (Table 111). Reconstitution of Enzymatic Activity-Enzymatic activities of purified P450H17mod(Hi~)~ are summarized in Table 111. The 17a-hydroxylase activities are almost the samefor progesterone and pregnenolone, which have similar K,,,values (Fig. 7A). Compared with reported valuesof 17,20-lyase activities in pig P450c17 (Nakajin et al., 1984; Suhara et al., 1984) or guinea pig P450c17 (Kominami et al., 19821, human P450c17 has a very low level of this activity (Fig. 7B). 17,20-Lyase activity using 170s-preg asa substrate was only 0.06 nmol/min/nmol P450, and undetectable using 170H-progas a substrate, even though 170H-progproduced a typical type I substrate binding spectrum. To determine whether thelow level of 17,204yase activity is attributed to the additionof 4 histidine residues at the COOH terminus, we comparedthe activities of P450H17mod (without histidinetail), P450Hl7mod(His), (withhistidinetail),and P450P17mod (pig P450c17 without histidine tail butmodification I at the NH2 terminus), each inisolated E. coli membrane fractions. As shown in Table lV, all threeenzymes showed similar turnover numbers for l7a-hydroxylation of progesterone but different 17,20-lyase activities. P450P17mod had a higher 17,20-lyase turnovernumberthaneither P450H17mod or P450H17rnod(His), andtheratio of hydroxylasellyase for P450P17mod is 3.0, which is very close to the reported value (Nakajin etal., 1984; Kominami et al.,1982). P450H17mod and P450H17m0d(His)~both have low turnover numbersfor 17,20lyase with no significant difference between the two, indicating that the addition of 4 histidine residuesat the COOH terminus does not appear toaffect this enzymatic activity.

0.6

,-

"_ ..F

Expression ofpCWHl7modVBl and VB2 and inVivo Enzyme Assays-pCWHl7modVBl and pCWHl7modVB2 were exas pCWHl7mod in E. coli. Neither pressed in the same manner mutant led to detectable 448nm peaks in CO difference spectra ineither whole bacteria or membrane fractions (datanot shown) although immunoreactive protein wasdetected in both and not in thecytosolic fractions. However, the expression levels of mutant immunoreactive protein were significantly less than pCWHl7mod (data not shown). Enzymatic activities of these mutants were measured by in vivo incubation of whole bacteria. As shown in Table V, P450H17modVBl demonstrated l7a-hydroxylase activity of approximately 15% of the P450H17mod, while P450H17modVB2 had no activity. Purification of P450H1 7modVB1(Hisl4-To investigate P 4 5 0 H 1 7 m o d V B li nm o r ed e t a i l ,w ec o n s t r u c t e d pCWH17modVBl(Hi~)~, expressed the mutant P450 in E. coli, and purified P450H17modVBl(Hi~)~ from a 1.5-liter culture. Although a CO difference spectrum was not obtained in whole bacteria, thepurified VB1 sample showed the expected 448 nm peak. From 1.5 liter of culture, we recovered 0.8 nmol of P450, having a 420 nm peak twice as intenseas the 448 nm peak in the CO difference spectrum (data not shown), having an estimated specific content 0.6 nmol/mg protein. 17a-Hydroxylase activity wasmeasured ina reconstitution system, and thisVB1 mutant showed very low 17a-hydroxylase activity(0.12 nmol of 170H-prog/min/nmol of P450). DISCUSSION

Standard purification procedures for microsomal forms of P450 employ conventional column systems, such as DEAEcellulose, DEAE-Sepharose, CM-Sepharose, hydroxylapatite, TABLEI11 Catalytic activitiesof purified P45OH17nwd(His), Values arethe average of three (17a-hydroxylase)or five (17,ZO-lyase) determinations for each substrate and presented as mean f S.D.ND, not detectable (less than 0.001). V,,

Substrate

17a-Hydroxylase

17,20-Lyase

nmol productlminlmmol P450

Pregnenolone l7a-OH-Reg Progesterone 17a-OH-F'rog

8.0 * 1.8

6.5 * 0.71

0.060 0.021

ND

K"I

K.

Pf

w

7.7 f 0.43 1.7 2.0f 1.2 6.3 f 0.88

1.6 1.4 5.3

ox

-Red Red-CO

- 0.06 AA

- 0.04

VnM) FIG.6. Spectral properties of purified P4KOH17mod(His),.Absolute absorption spectra (left)and CO difference spectra (right).The cuvette contained 4.6 nmoW450 ml of buffer (0.1 M sodium phosphate, pH 7.2, 20% glycerol,0.02%Emulgen 913, 50 PM progesterone) for the absolute absorption spectrum and2.3 nmoW450 ml in the same buffer for the difference spectrum.Ox, oxidized form;Red, reduced with sodium dithionite; Red-CO, reduced form coordinated with CO.

P450cl7 in Expression and Purification of Human A

11s (pM-5

B h

0

2l n

120, 100.