Expression of a cDNA encoding a rat liver glutathione S-transferase Ya subunit in Escherichia coli

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 269, No. 2, March, pp. 536-543,1989

Expression of a cDNA Encoding a Rat Liver Glutathione S-Transferase Subunit in fscherichia co/i REGINA

W. WANG*,’

CECIL B. PICKETT,?

AND

ANTHONY

Y. H. LU*

*Department of Animal & Exploratory LIrug Metabolism and fDepartment of Molecular Pharmacology Biochemistq, Merck Sharp & Dohme Research Laboratories, Rahway, New Jersey 07065 Received September 13,1988, and in revised form November

Ya

and

3,1988

A full length cDNA clone, pGTB38 (C. B. Pickett et al. (1984) J. Biol. Chem. 259,51825188), complementary to a rat liver glutathione S-transferase Ya mRNA has been expressed in Escherichia coli. The cDNA insert was isolated from pGTB38 using Mae1 endonuclease digestion and was inserted into the expression vector pKK2.7 under the control of the tat promoter. Upon transformation of the expression vector into E. coli, two protein bands with molecular weights lower than the full-length Ya subunit were detected by Western blot analysis in the cell lysate of E. coli. These lower-molecular-weight proteins most likely result from incorrect initiation of translation at internal AUG codons instead of the first AUG codon of the mRNA. In order to eliminate the problem of incorrect initiation, the glutathione S-transferase Ya cDNA was isolated from the expression vector and digested with Ba131 to remove extra nucleotides from the 5’ noncoding region. The protein expressed by this expression plasmid, pKK-GTB34, comigrated with the Ya subunit on sodium dodecyl sulfate polyacrylamide gels and was recognized by antibodies against the YaYc heterodimer. The expressed Ya homodimer was purified by S-hexylglutathione affinity and ion-exchange chromatographies. Approximately 50 mg pure protein was obtained from 9 liters of E. coli culture. The expressed Ya homodimer displayed glutathione-conjugating, peroxidase, and isomerase activities, which are identical to those of the native enzyme purified from rat liver cytosol. Protein sequencing indicates that the expressed protein has a serine as the NH2 terminus whereas the NH2 terminus of the glutathione S-transferase Ya homodimer purified from rat liver cystosol is apparently blocked. o 1989Academie PCMS. IIK.

The cytosolic glutathione S-transferases are a family of isozymes which catalyze the conjugation of glutathione to a wide variety of electrophilic substrates (l-3). To date, a number of glutathione S-transferases have been purified from various animal species and characterized to various extents (2,3). All isozymes are either heterodimers or homodimers composed of various subunits.2 Recently, cDNA clones 1 To whom correspondence should be addressed at: Animal & Exploratory Drug Metabolism, Building 80, Room A12, Merck Sharp & Dohme Research Laboratories, P.O. Box 2000, Rahway, NJ 07065. ‘Under the proposed nomenclature (4), subunits 1, 2, 3, 4, 6, 7, and 8 are equivalent to subunits Ya, Ye, 0003-9861/89 $3.00 Copyright All rights

0 1989 by Academic Press, Inc. of reproduction in any form reserved.

complementary to the mRNAs specific for the Ya, Yc, and other subunits have been constructed, and the deduced amino acid sequences for these subunits have been reported (5-20). With these cDNA probes, Ya, Yc, Ybl, and Yb2 mRNAs have been shown to be differentially regulated by xenobiotics (6,21). Although considerable progress has been made in understanding the molecular mechanisms that regulate glutathione Stransferase gene expression, information regarding the structure and function of various glutathione S-transferase isoYbl, Yb2, Yn, Yp, and Yk, respectively. Yc are used in this manuscript. 536

Terms Ya and

RAT

GLUTATHIONE

S-TRANSFERASE

zymes is still lacking. Apparently homogeneous glutathione S-transferase preparations isolated from animals may contain more than one structurally related subunit. The microheterogeneity in glutathione S-transferase subunits may complicate structural and functional analysis of forms purified from rat liver (6,8,11,12,19, 22). For example, three rat liver Ya cDNA clones, differing by only a few nucleotides, have been isolated and characterized (6,11, 19). Wang et al. (23) also reported the presence of multiple Ya subunits in rats based on monoclonal antibody studies. This microheterogeneity of the Ya subunits cannot be detected in an enzyme preparation by either SDS3- gel electrophoresis or peptide mapping (24). On the other hand, the expression of a cDNA encoding a specific glutathione S-transferase subunit in Escherichia coli or yeast not only would produce a large quantity of enzyme for structural study but also would ensure the production of a dimer containing a specific glutathione S-transferase subunit. Recently, a rat glutathione S-transferase and a human enzyme were expressed in E. coli (25, 26). In this paper, the expression in E. coli of the cDNA clone pGTB38 (6), which is complementary to a rat liver glutathione S-transferase Ya mRNA, is described. MATERIALS

AND

METHODS

Cbnstruction of the expressimz. plnsmid. The construction of the expression plasmid pKK-GTB34 is diagrammed in Fig. 1. Clone pGTB38 (6), containing the full-length cDNA complementary to a rat liver glutathione Ya mRNA, was digested with Mae1 (Boehringer-Mannheim) and the fragments were separated by electrophoresis on a 1% agarose gel. The nilae1 fragment which contains the coding nucleotide sequence was isolated from the gel and treated with the Klenow fragment of DNA polymerase I (IBI) to fill in the sticky ends. The plasmid pKK-GTB17 was obtained by insertion of the Mae1 fragment into the SmaI site of the dephosphorylated expression vector pKK2.7 which was prepared by digesting pKK223-3 (Pharmacia) with NurI and NdeI to generate a 2700bp vector containing a single S&I site (27).

3 Abbreviations used: SDS, sodium dodecyl sulfate; IPTG, isopropyl P-D-thiogalactopyranoside; bp, base pairs; CDNB, 1-chloro-2,4-dinitrobenzene.

Ya cDNA

IN E. coli

537

The Mae1 fragment was released from pKK-GTB17 by Sol1 and EcoRI (BRL) and digested with a “slow” form B&l nuclease (IBI) for 6 min at 3O”C, then subsequently blunt-ended. The B&.31-treated cDNA was ligated into the SmaI site of the dephosphorylated expression vector pKK2.7 with T4 DNA ligase to construct the expression plasmid pKK-GTB34. The plasmid was purified by standard techniques (28,29) and the orientation of the inserted Ya cDNA in plasmid was determined by Hind111 (BRL) digestion and DNA sequence analysis. Bacterial transformation and cell growth. The expression vector pKK2.7 and the constructed plasmids were used to transform E. coli strains JM 105 (the lac io host, Pharmacia) and AB 1899 (the Ion-l proteasedeficient strain, E. coli Genetic Stock Center, Yale University) by the calcium chloride procedure (30, 31). Colonies were selected by ampicillin resistance and screened by colony hybridization (32) with a 32Plabeled probe which was prepared by nick translation (33) of the corresponding cDNA fragment. The transformants were grown at 37°C in LB broth containing 100 fig/ml ampicillin. When the JM 105 strain was used for transformation, 1 mM IPTG (BRL) was added to the medium when the AssOof the culture reached 0.4. Cells were harvested at the stationary phase. No IPTG was needed when strain AB 1899 was used for transformation. Pu@kation of the expressed Ya subunit. Cells from 9 liters of pKK-GTB34 transformed E. coli AB 1899 overnight culture were harvested by centrifugation at 10,OOOgfor 20 min, and disrupted by sonication in 10 mM NaP04, pH 7.4, containing 5 mM EDTA. The cell debris and unbroken cells were removed after centrifugation at 10,OOOgfor 25 min and the resulting supernatant was centrifuged at 100,OOOgfor 60 min. The cytosolic fraction (100,OOOg supernatant) was concentrated using a PM-10 membrane and loaded onto a Sephadex G-50 column. The fractions with transferase activities were pooled and purified by S-hexylglutathione-linked Sepharose 6B affinity and CM-cellulose column chromatographies as described (34).

PutiJcation

of rut liver glutathiwze S-transferuse.

Rat liver glutathione S-transferases YaYc and YaYa were used as reference standards. They were purified from liver cytosol of a phenobarbital-induced rat using a glutathione-linked Sepharose 6B affinity column (35) followed by ion-exchange chromatography (36). Western blot analysis. Proteins in E. coli lysate or in various fractions during purification were separated on 12.5% SDS polyacrylamide gels by the procedure of Laemmli (37), and then transferred to nitrocellulose sheets (Schleicher & Schuell) using the method described by Towbin et al. (38). The nitrocellulose sheets were treated with blocking buffer (phosphatebuffered saline containing 3% nonfat dry milk and 0.05% Tween 20) for 2 h at room temperature. The sheets were incubated with a 1:500 dilution of a mono-

538

WANG, MaeI.

PICKETT,

AND

LU

pst I

‘Q

Tetr

I

“” Ig

Mae 1 Digestion Fill in with Klenow

Promoter

I

Smal Digestion

rrn B 1

Promoter

SalI-EcoRI

I

Digestion

I

Bal31 Digestion Fill in with Klenow

1

SmaI Digestion

Ligation

rrnB Terminators

FIG. 1. Construction procedure of the expression plasmid pKK-GTB34. The cDNA was digested and inserted into vector pKK2.7 as described in the text. The hatched boxes indicate the inserted Ya cDNA. The solid boxes indicate the promotor and terminator regions. Amp’ and Tet’ designate the ampicillin-resistance and tetracycline-resistance genes, respectively.

clonal antibody which is specific for the Ya subunit (23), or polyclonal antibodies against YaYc subunits (6). After the sheets were washed with blocking buffer, they were treated with 2 ).&i ‘a51-Protein A (30 mCi/mg, Amersham Corp.) in 20 ml of blocking buffer for an additional hour followed by washing with blocking buffer and phosphate-buffered saline. The sheets were air-dried and autoradiographed. DNA sequence analysis. Plasmid DNA was prepared for DNA sequencing by adding NaCl to a concentration of 0.8 M and incubating with an equal volume of 13% polyethylene glycol at 4°C for 20 min, then ethanol-precipitated and resuspended in 10 mM

Tris-HCl, pH 8.0, containing 1 mM EDTA. The synthetic oligonucleotide primer, which contained a 20nucleotide sequence of the promoter region of the expression vector pKK2.7, was synthesized on a Biosearch 8700 synthesizer (Biosearch) and purified on an oligonucleotide purification cartridge (Applied Biosystems). This universal primer, was denatured with 0.2 M NaOH, 0.2 mM EDTA, precipitated with ethanol, and subsequently annealed to plasmid DNA. DNA sequencing was performed with the modified dideoxy chain termination technique (39) using bacteriophage T7 DNA polymerase (40) as described in the Sequenase kit (United States Biochemical Corp.).

RAT

GLUTATHIONE

S-TRANSFERASE

414,400

FIG. 2. Western blot analysis of cell lysates of E. wli strain AB 1899. Lane 1, E. coli containing vector pKK2.7; lane 2, E. coli containing pKK-GRB17; lane 3, E. coli containing pKK-GTB34; lane 4, rat liver YaYa and cell lysates of E. coli containing pKK-GTB17; lane 5, rat liver YaYa. Proteins are separated on a SDS polyacrylamide gel and transferred to nitrocellulose, then immunoblotted using polyclonal antibodies against YaYc.

Amino acid sequence analysis. One milligram of the purified expressed Ya subunit was sequenced with an Applied Biosystems 470A protein sequencer which was interfaced with the Applied Biosystems 120A analyzer (41). Assays. Glutathione Stransferase activities with different substrates were measured spectrophotometrically at 25°C according to published procedures. The assay conditions with various substrates, lchloro-2,4-dinitrobenzene (Sigma), 1,2-dichloro-4-nitrobenzene (Sigma), ethacrynic acid (Sigma), p-nitrobenzyl chloride (Aldrich), 1,2-epoxy-3-(p-nitrophenoxy)propane (Sigma) and bromosulfophthalein (Sigma), were adapted from Habig et al. (42) and Habig and Jakoby (43). Glutathione peroxidase activity with cumene hydroperoxide (Matheson Coleman & Bell) as the substrate and isomerase activity with A’-androstene-3,17-dione (Steraloids, Inc.) as substrate were determined as de,cribed (44-46). Protein concentrations were measured by the method of Lowry ef al. (47) with bovine serum albumin as standard. RESULTS

AND

Plasmid Construction in E. coli

DISCUSSION

and Its Exp-essicm

The Mae1 fragment of pGTB38, consisting of the complete coding region, 25 bp of

Ya cDNA

539

IN E. coli

the 5’ noncoding region, 114 bp of the 3’ noncoding region, 30 bp of poly(A), and 51 bp of pBR322 sequence, was ligated into the expression vector pKK2.7 between the tat promoter and the rrnB ribosomal RNA transcription terminator. The presence of this fragment in the vector was confirmed by colony hybridization and DNA sequence analysis. When plasmid pKK-GTB1’7 was transformed into E. coli strains JM 105 or AB 1899, two proteins of lower molecular weight than the transferase Ya subunit were detected by Western blot analysis with antisera against YaYc (Fig. 2, lane 2), but little or no enzyme activity could be detected in the cell lysate of the transformed E. coli (Fig. 3, curve 2). To test whether the E. coli lysate contained a protease capable of degrading the full length Ya subunit into smaller fragments, a purified rat liver YaYa sample was first incubated with the cell lysate and then subjected to Western blot analysis. No apparent degradation of the rat liver YaYa sample was observed (Fig. 2, lane 4). These results indicate that the expression of the truncated Ya subunit is probably not due to protease cleavage of

0.4 -

0.0 0

I 0.5

I 1.0

I 1.5

I 2.0

Ttme (min)

FIG. 3. Glutathione Stransferase activity in E. coli strain AB 1899 cell lysates. The E. coli cells of overnight culture were disrupted by sonication and the enzyme activities of the cell lysates were determined with CDNB as substrate. Curve 1, E. coli containing vector pKK2.7; curve 2, E. coli containing pKKGTB17; curve 3, E. coli containing pKK-GTB34.

540

WANG,

PICKETT,

Ya cDNA

LU

transformed into E. coli strain JM 105, the cells produced full-length glutathione Stransferase Ya subunit after IPTG induction. This protein could also be expressed to the same level without IPTG induction in E. coli strain AB 1899 containing plasmid pKK-GTB34. The expressed protein can be recognized by the YaYc polyclonal antibodies (Fig. 2, lane 3) and displayed glutathione-conjugating activity using CDNB as substrate (Fig. 3, curve 3).

S/D

5’ ACA&&ACAGAAGCTATGTCTGGG Vector

AND

3

Ya cDNA

FIG. 4. Diagrammatic representation of the expression plasmid pKK-GTB34. The segment labeled Ya cDNA was ligated into vector pKK2.7 between the tat promoter and the rrnB ribosomal RNA transcription terminator region. The nucleotide sequence of the junction between vector pKK2.7 and Ya cDNA is shown at the bottom. The nucleotides from the vector are underlined. S/D indicates the Shine-Dalgarno sequence.

the full-length transferase Ya in E. coli. Control experiments showed the absence of antibody-reactive proteins in E. coli cells containing vector pKK2.7 alone (Fig. 2, lane 1). Therefore, these results suggest that the expression of truncated proteins may be due to the initiation of translation at internal AUG codons of the Ya mRNA. In an attempt to eliminate the problem of incorrect initiation, the Mae1 fragment was isolated from pKK-GTB17 and digested with Ba131 nuclease to remove nucleotides from the 5’untranslated region in order to position the Shine-Dalgarno sequence closer to the initiation codon and/ or to avoid the possible formation of potential secondary structure around the initiation codon. After the Bal31 digestion, the nucleotide sequence of the junction area between the vector and cDNA was determined. Only 3 bp of the 5’ untranslated region of the cDNA were retained after Bal31 cleavage. Also, it was found that 5 bp around the EcoRI site of the polylinker was deleted during SmaI digestion to open the vector. The initiation codon in this construction is located 11 nucleotides downstream of the Shine-Dalgarno sequence (Fig. 4). When plasmid pKK-GTB34 was

PuriJication and Characterization Expressed Yu Subunit

of the

Cells from pKK-GTB34 transformed E. coli AB 1899 were used for purification of the glutathione S-transferase Ya homodimer. After being passed through the Sephadex G-50 column, the cytosolic fraction was loaded onto an S-hexylglutathione affinity column. A distinct protein peak containing most of the glutathione transferase activity was eluted by buffer containing glutathione and S-hexylglutathione (Fig. 5), and the pooled enzyme was further purified on a CM-cellulose column. Starting from 9 liters of E. coli culture, approximately 50 mg of purified YaYa was obtained with this three-step purification procedure (Table I). Samples from each step were run on 12.5% SDS polyacrylamide gels and the

T

FIG. 5. Purification of the expressed Ya homodimer by 9hexylglutathione-linked Sepharose 6B affinity chromatography. After application of the sample from the Sephadex G-50 column, the affinity matrix was washed with 25 mM Tris-HCl, pH 8.0, containing 0.2 M KC1 until no absorption at 280 nm could be detected in the effluent. The retained protein was eluted with 25 mM Tris-HC1 containing 0.2 M KCl, 5 mM Shexylglutathione, and 2.5 mM glutathione. The enzyme activity was assayed with CDNB as substrate.

RAT

GLUTATHIONE

S-TRANSFERASE TABLE

Ya cDNA

541

IN E. coli

I

PURIFICATION OF THE EXPRESSED GLUTHATHIONE S-TRANSFERASE YaYa FROM E. coli

Purification

step

Cytosolic fraction Sephadex column Affinity column CM-cellulose column u Activity

was determined

Total activity” (pmol/min) 4524 4441 2697 2542

Yield (%I

Specific activity (*mol/min/mg)

Total protein bud

0.63 0.67 44 52

7178 6607 62 49

100 98 60 56

with CDNB as substrate.

proteins were detected either by fluorescent visualization directly on a uv transilluminator without staining (48) or by Coomassie blue staining (Fig. 6A). The purified protein from the affinity column and the CM column showed a single band on the gels that comigrated with the purified rat liver Ya subunit. Two sets of gels with the same samples were immunoblotted with anti-YaYc polyclonal antibodies and an anti-Ya monoclonal antibody. A single band of immunoreactive protein was detected in all samples throughout the purification steps (Figs. 6B and 6C). Thus, the Western blot analysis revealed that the expressed protein had the same subunit molecular weight as the purified rat liver Ya subunit and could be recognized by antibody against the Ya subunit. The amino acid sequence of the Ya subunit of either the Ya homodimer or YaYc

heterodimer purified from rat liver cytosol has never been determined by conventional protein sequencing since the NH2 termini of the isolated enzymes are blocked (7). In order to determine the NH2terminal sequence of the expressed Ya subunit, the purified protein was subjected to protein sequencing analysis. Figure 7 shows that the sequence of the first 30 NH2-terminal amino acid residues of the expressed glutathione S-transferase Ya subunit matches the sequence deduced from the cDNA sequence (6). Sequence analysis also showed that 90% of the expressed protein had a serine NH2 terminus and that methionine had been effectively removed by the E. coli methionine aminopeptidase (49). However, approximately 10% of the expressed Ya subunit still retained methionine, possibly due to the overproduction of the Ya subunit in E. coli resulting in the incomplete processing of the methionine residue.

A. B.

FIG. 6. Western blot analysis of the expressed Ya homodimer from various stages of purification. (A) SDS polyacrylamide gel stained with Coomasie brilliant blue. Lane 1, cytosolie fraction; lane 2, affinity column fraction; lane 3, CM column fraction; lane 4, rat liver YaYa; lane 5, rat liver YaYc. SDS polyacrylamide gels identical to that of A were transferred to nitrocellulose and immunoblotted with polyclonal antibodies against YaYc (B); or immunoblotted with monoclonal antibody against Ya subunit (C).

ATG MET

Ser

Gly

Phe

Asn

TCT se1

GGG AAG CCA GTG CTT Gly LY.3 PI0 "al Leu

Lys

CAC TAC HIS Tyr

TX Phe

AAT AS"

Ala

Arg

Cys

Arg

Trp

Gly

Pro

Arg

Val

Met

Leu

Giu

His

Tyr

Ile

GCC CGG GGC AGA AK Ala Arg Gly Arg Met

GAG TGC AK Glu Cys Ile

CGG TGG Rrg Trp

Leu

Gly

Phe

Leu

Ala

Ala

Ala

"al

Glu

CTC CTG GCT GCR GCA GGA GTG GAG TTT Leu Leu Ala Ala Ala Gly "al Glu Phe

Glu GAA Glu

FIG. 7. NH,-terminal amino acid sequence of the expressed Ya homodimer. The sequence of the first 30 NH,-terminal amino acid residues determined from protein sequencing (A) is compared with the amino acid sequence deduced from the cDNA nucleotide sequence (B).

542

WANG, TABLE

PICKETT,

II

SUBSTRATE SPECIFICITY OF THE EXPRESSED GLUTATHIONE S-TRANSFERASE YaYa ISOZYME Relative specific activity (%)a

Substrate l-Chloro-2,4-dinitrobenzene 1,2-Dichloro-4-nitrobenzene Bromosulfophtbalein Ethacrynic acid Cumene hydroperoxide A5-Androstene-3,17-dione pNitrobenzy1 chloride 1,2-Epoxy-3-(p-nitrophenoxy)propane

Expressed YaYa

Rat liver YaYa

100 0.12
100 0.11 to.01 0.19 4.36 3.94 0.25



a Values are given as percentages of the specific activities determined with CDNB as substrate (expressed YaYa, 52 rmol/min/mg; rat liver YaYa, 36 pmol/min/mg).

The substrate specificities of the expressed protein and the purified rat liver Ya homodimer are compared in Table II. When CDNB was used as the substrate, the specific activities (pmol/min/mg protein) were 52 for the expressed protein and 36 for the purified rat liver YaYa. With other substrates, the relative activities for these two enzymes were virtually identical. As expected, the expressed enzyme had high isomerase activity with A5-androstene3,17-dione as substrate since the purified rat liver transferase YaYa had been reported to have the highest isomerase activity among all the rat transferases so far characterized (50). In summary, a cDNA encoding a rat liver glutathione S-transferase Ya subunit has been expressed in E. coli in high yield. The expressed glutathione S-transferase Ya homodimer displays glutathione-conjugating, isomerase, and peroxidase activities. Also, the expressed protein is apparently dimeric since it coelutes with the purified rat liver Ya homodimer on a molecular sieving column (Pharmacia Superose 12 HR 10/30) in a FPLC system (data not shown). The ability to express

AND

LU

and purify to homogeneity a glutathione Stransferase subunit should facilitate a detailed structure-function analysis of this glutathione S-transferase protein using site-directed mutagenesis and X-ray crystallographic techniques. ACKNOWLEDGMENTS We thank Dr. David Linemeyer for valuable discussions and for providing us vector pKK2.7, Dr. Irene Wang for the use of monoclonal antibody, Mr. Joseph Wu for running the Superose 12 column, and Mrs. Terry Rafferty for her assistance in the preparation of this manuscript. REFERENCES 1. JAKOBY, W. B. (1978) Adv. EnzymoL Relat. Areas. Mol. BioL 46,383-414. 2. MANNERVIK, B. (1985) Adv. EnzymoL Relat. Areas Mol. BioL 57,357-417. 3. MANNERVIK, B., AND DANIELSON, U. H. (1988) CRC &it. Rev. B&hem. 23,283-337. 4. JAKOBY, W. B., KETTERER, B., AND MANNERVIK, B. (1984) Biochem. Pharmacol. 33,2539-2540. 5. ABRAMOVITZ, M., AND LISTOWSKY, I. (1987) J. BioL Chem 262,7770-7773. 6. PICKETT, C. B., TELAKOWSKI-HOPKINS, C. A., DING, G. J.-F., ARGENBRIGHT, L., AND Lu, A. Y. H. (1984) J. BioL Chem. 259,5182-5188. 7. TELAKOWSKI-HOPKINS, C. A., RODKEY, J. A.,BENNETT, C. D., Lu, A. Y. H., AND PICKETT, C. B. (1985) J. Biol Chem. 260,5820-5825. 8. DING G. J.-F., Lu, A. Y. H., AND PICKETT, C. B. (1985) J. BioL Chem. 260,13268-13271. 9. DING, G. J.-F., DING, V. D.-H., RODKEY, J. A., BENNETT, C. D., Lu, A. Y. H., AND PICKETT, C. B. (1986) J. BioL Chem. 261,7952-7957. 10. Tu, C.-P. D., WEISS, M. J., KARAKAWA, W. W., AND REDDY, C. C. (1982) Nucleic Acids Res. 10,54075419. 11. LAI, H. C. J., LI, N., WEISS, M. J., REDDY, C. C., AND Tu, C.-P. D. (1984) J. BioL Chem. 259,55365542. 12. LAI, H. C.-J., GROVE, G., and Tu, C.-P. D. (1986) Nucleic Acids Res. 14,6101-6114. 13. LAI, H. C.-J., AND Tu, C.-P. D. (1976)J. BioL Chem. 261,13793-13799. 14. Tu, C.-P. D., AND &IAN, B. (1986) B&hem Biophys. Res. Commun. 141,229-237. 15. RHOADS, D. M., ZARLENGO, R. P., AND Tu, C.-P. D. (1987) Biochem. Biophys. Res. Cornmun. 145,474-481. 16. TAYLOR, J. B., CRAIG, R. K., BEALE, D., AND KETTERER, B. (1984) Biochem. J 219,223-231. 17. SUGUOKO, Y., KANO, T., OKUDA, A., SAKAI, M., KITAGAWA, T., AND MURAMATSU, M. (1985) Nucleic Acids Res. 13,6049-6057.

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S-TRANSFERASE

18. PEMBLE, S. E., TAYLOR, J. B., AND KETTERER, B. (1986) Biochem. J. 240,885~889. 19. ROTHKOPF, G. S., TELAKOWSKI-HOPKINS, C. A., STOTISH, R. L., AND PICKETT, C. B. (1986) Bicchemistry 25,993-1002. 20. BUTERA, L., MONNIER, J. R., CAMPBELL, E., AND BHARGAVA, M. M. (1987) Biochem. Biophvs. Res. Commun. 142,986-992. 21. DING, V. D.-H., AND PICKETT, C. B. (1985) Arch. Biochem. Biophys. 240,553-559. 22. CHOW, N.-W. I., WHANG-PENG, J., KAO-SHAN, C.-S., TAM, M. F., LAI, H.-C. J., AND Tu, C.-P. D. (1988) J. BioL Chem. 263,12797-12800. 23. WANG, I. Y., TUNG, E., WANG, A., ARGENBRIGHT, L., WANG, R. W., PICKETT, C. B., AND Lu, A. Y. H. (1986) Arch. Biochem. Biophys. 245, 543-547. 24. PICKETT, C. B., TELAKOWSKI-HOPKINS, C. A., DING, G. J.-F., WANG, R. W., AND Lu, A. Y. H. (1985) in Microsome and Drug Oxidations (Boobis, A. R., Caldwell, J., DeMatteis, F., and Elcombe, C. R., Eds.), pp. 128-135. Taylor & Francis, London. 25. LAI, H.-C. J., &IAN, B., AND Tu, C.-P. D. (1987)Fed. Proc. 46,2186. 26. BOARD, P. G., AND PIERCE, K., (1987) Biochem. J. 248,937-941. 27. LINEMEYER, D. L., KELLY, L. J., MENKE, J. G., GIMENEZ-GALLEGO, G., DISALVO, J., AND THOMAS, K. A. (1987) Biotechnology 5,960-964. 28. CLEWELL, D. B., AND HELINSKI, D. R. (1969) Proc. Natl. Acud. Sci. USA 62,1159-1166. 29. CLEWELL, D. B. (1972) .I BacterioL 110,667-676. 30. MANDEL, M., AND HIGA, A. (1970) J. Mol. BioL 53, 159-162. 31. COHEN, S. N., CHANG, A. C. Y., AND Hsu, L. (1972) Proc. NatL Acad Sci USA 69,2110-2114. 32. MANIATIS, T., FRITSCH, E. F., AND SAMBROOK, J. (1982) in Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 33. RIGBY, P. W. J., DIECKMANN, M., RHODES, C., AND BERG, P. (1977) J. Mol. Biol. 113,237-251.

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IN E. coli

543

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