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[19] P r o d u c t i o n o f Site-Specific P 4 5 0 A n t i b o d i e s Using R e c o m b i n a n t F u s i o n P r o t e i n s as A n t i g e n s B y THOMAS FRIEDBERG, W O L F G A N G KISSEL, MICHAEL A R A N D ,

and FRANZ OESCH Introduction The high degree of homology among members of some cytochrome P450 families has complicated studies on the expression of individual P450 cytochromes. Specific oligonucleotide probes and monoclonal antibodies have been developed that can discriminate between cytochrome P450 mRNAs L2and proteins, respectively.~ However, generating monoclonal antibodies which are monospecific for a member of a cytochrome P450 subfamily requires the screening and characterization of a large number of positive hybridomas. A more straightforward approach for the development of P450 form-specific antibodies involves preparation of antibodies against defined variable peptide segments in the individual members of a P450 subfamily. The antigens used for this approach can be chemically synthesized peptides coupled to carrier proteins 7 or fusion proteins composed of a linear sequence of a P450 unrelated protein and a P450 peptide.8 The large-scale production of these proteins is achieved in bacteria harboring an expression vector directing the synthesis of the fusion protein. The DNA sequence encoding the P450 peptide is usually a P450 cDNA restriction fragment. The fragment has to be inserted into the parental expression vector which codes for the P450 unrelated protein in such a way that both sequences maintain their original translation reading frame. Various prokaryotic expression vectors for the production of fusion l C. M. Giachelli and G. J. Omiecinski, Mol. Pharmacol. 31, 477 (1987). 2 C. J. Omiecinski, Nucleic Acids Res. 14, 1525 (1986). 3 L. M. Reik, W. Levin, D. E. Ryan, S. L. Maines, and P. E. Thomas, Arch. Biochem. Biophys. 242, 365 (1985). 4 S. S. Park, T. Fujino, D. West, F. P. Guengerich, and H. V. Gelboin, Cancer Res. 42, 1798 (1982). 5 S. S. Park, T. Fujino, H. Miller, F. P. Guengerich, and H. V. Gelboin, Biochem, Pharmacol. 33, 2071 (1984). 6 S. S. Park, D. J. Waxman, H. Miller, R. Robinson, C. Attisano, F. P. Guengerich, and H. V. Gelboin, Biochem. Pharmacol. 35, 2859 (1986). 7 A. B. Frey, D. J. Waxman, and G. Kreibich, J. Biol. Chem. 2,60, 15253 (1985). 8 F. Oesch, D. J. Waxman, J. J. Morrissey, W. Honscha, W. Kissel, and T. Friedberg, Arch. Biochem. Biophys. 270, 23 (1989).

METHODS IN ENZYMOLOGY, VOL. 206

Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

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proteins have been described. These expression vectors encode proteins like MS2 polymerase,9 dihydrofolate reductase,10 protein A, H and glutathione S-transferase.12 Others report the use and the construction of expression vectors in which the eDNA fragments coding for the peptide of interest are linked to sequences coding for the carboxy terminus of fl-galactosidase13 or to a/3-galactosidase containing some sequences derived from the cro and the l a c I gene at its amino terminus.14 The location of the subcloned eDNA fragment at the 3' end of the sequence coding for /3-galactosidase ensures that the expression of this construct is relatively independent of the inserted eDNA sequence. Compared to many other prokaryotic expression systems for fusion proteins, the systems described by Riither and Miiller HilP 3 and by Stanley and Luzio TM have the distinct advantage of containing a multiple cloning site that allows fusions in each of the three reading frames, thus facilitating the subcloning of cDNAs in the correct reading frame. In this report we describe the generation of P450-peptide fusion proteins using the plasmids pEX1, pEX2, and pEX3 described by Stanley and Luzio. ~4 These vectors are available commercially (Clontech Laboratories, Inc., Palo Alto, CA). Principles of Method Figure 1 shows a map of pEX2 and the organization of the c r o - l a c Z gene. The other two versions o f p E X were generated by frameshifts of + 1 and - 1 at the E c o R I site of the polylinker. Thus, this restriction site is not available in pEX1 and pEX3. Recently derivatives of pEX2, namely, pEX11, pEXI2, and pEX13, have been constructed with an extended multiple cloning site. ~5 The host bacterium for the pEX vectors is the E s c h e r i c h i a coli strain pop 2136, which carries on its genome a defective prophage coding for the ci857ts element of phage h. When the host bacterium is grown at 30° it synthesizes the cI repressor protein. This protein suppresses the kP R promoter on pEX (Fig. 1) and therefore inhibits the synthesis of fusion protein. Shifting the temperature to 42° leads to a degradation of the ci857ts suppressor, resulting in the activation of the extremely powerful kPR promoter. In addition the expression of the cro repressor gene of the prophage inhibits the expression of bacterial genes. 9 K. Strebel, E. Beck, K. Strohmaier,and H. Schaller, J. Virol. $7, 983 (1986). 10p. S. Vermersch, M. R. Klass, and G. N. Bennett, Gene 41, 289 (1986). It B. Nilsson, L. Abrahamsen, and M. Uhlen, EMBO J. 4, 1075(1985). 12D. B. Smith and K. S. Johnson, Gene 67, 31 (1988). 13U. Rfitherand B. MOilerHill, EMBO J. 2, 1791 (1983). 14K. K. Stanleyand J. P. Luzio, EMBO J. 3, 1429(1984). 15j. G. Kusters,E. J. Jager, and B. A. M. van der Zeijst,Nucleic Acids Res. 17, 8007(1989).

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PRODUCTION OF SITE-SPECIFIC P450 ANTIBODIES a

195

MRtII

EcoRV

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Smol EcoRl b PR CrO'bcl'

(oct

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FIG. 1. Map of pEX2. Unique restriction sites are shown in (a), the organization of the cro-lacZ gene in (b). STOP, Stop codon in all three reading frames; Tfd, transcription

terminator fragment.

This cascade of events leads to the expression of a cro-#-galactosidase (cro-lacZ) hybrid protein which accounts for approximately 30% of the total extractable protein) 4 The fusion proteins are very insoluble and form inclusion bodies. These inclusion bodies are toxic for the host. Therefore, the production of fusion proteins is only induced by a temperature shift when the bacteria have been grown to an optimal density. The insolubility of the fusion protein facilitates its purification by detergent extraction and moreover protects it from proteolytic degradation. 16 The detergent-extracted fusion protein is further purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and is then used for immunization. The resulting antisera can be purified by affinity chromatography on a column containing the cro-lacZ protein to remove antibodies reactive toward the fusion partner. If necessary the antibodies can be further purified on a column containing the fusion protein. Antibodies recognizing the peptide sequence of interest are eluted at low pH. 16y . S. Cheng, D. Y. Kwoh, T. J. Kwoh, B. C. Solvedt, and D. Zipser, Gene 14, 121 (1981).

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Methods

Growth o f Bacteria and Generation o f Competent Cells The host strain E. coli pop 2136 is grown in LB medium at 37°. Escherichia coli pop 2136 cells containing the pEX vectors have to be grown below 34°. Only one transformation procedure is given below. This method is based on the Hanahan method 17'18and yields up to 2 x 10 7 transformants per microgram pEX.

Reagents SOB and SOC medium: 2% Bacto-tryptone (Difco, Detroit, MI), 0.5% Bacto yeast extract (Difco), 10 mM NaCl, 2.5 mM KC1, 10 mM MgCl 2 , 10 raM MgSO4. Autoclave tryptone, yeast extract, NaCI, and KC1 in the purest water available. Use within 2 weeks. Make a stock solution of 1 M of each Mg salt, sterile filter, and add just before use. SOC medium: SOB plus 20 mM glucose Dimethyl sulfoxide (DMSO) solution: 90% (v/v) DMSO (spectroscopy grade, fresh) in 10 mM potassium acetate, pH 7.5 FSB medium: 100 mM KCI ultrapure, 45 mM MnCI 2 • 4 H 2 0 , 10 mM C a C 1 2 , 3 mM hexamminecobalt chloride, 10 mM potassium acetate (from a 1 M potassium acetate stock solution, pH 7.5), 10% (v/v) redistilled glycerol. Adjust the solution with 0. I N HCI to pH 6.4. Sterile filter and store at 4°. Procedure. Streak a clump of frozen E. coli pop 2136 cells on an SOB plate and grow at 37°. Pick two colonies (2.5 mm diameter), combine in I ml SOB, and disperse by vortexing. Inoculate with this suspension a prerinsed 250-ml flask containing 25 ml SOB and grow the bacteria at 37° to a density of 0.15 OD600. Dilute the culture 1 : 1 in SOB and grow at 34° to an OD600 of 0.3. Transfer to a 50-ml Falcon tube (Becton Dickinson Labware, Lincoln Park, NY) and chill on ice for 15 min. Centrifuge for 20 min at 4000 g at 4 °. Resuspend the resulting pellet in FSB medium (1/3 volume of the culture volume). Leave on ice for 10 min. Recentrifuge as above and resuspend the pellet in FSB (1/12.5 volume of the original culture). Add DMSO solution to 3.5% and leave on ice for 5 min. Add a second aliquot of DMSO to 7% (v/v). The cells are quickly frozen in liquid nitrogen and stored at - 7 0 °. Temperature shifts in the freezer compartment should be avoided. For transformation with DNA, the competent cells are partially thawed 17 D. H. Hanahan, J. Mol. Biol. 166, 557 (1983). 18 H. Okayama, M. Kaiwichi, M. Brownstein, F. Lee, T. Yokota, and K. Arai, this series, Vol. 154, p. 23.

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at room temperature and completely thawed on ice. Aliquots (210/xl) of competent cells are transferred to 10-ml Falcon tubes placed in an ice bath. Maximally I0 ng of DNA is added, followed by incubation on ice for 30 min. The bacteria are then heat-shocked at 37° for 2 min, followed by incubation on ice for 2 min. Then 0.8 ml SOC (20°) is added, and the cells are shaken vigorously at 30° for 1 hr. The cells are plated on plates containing ampiciUin (for pEX) and grown at 30°. Plasmid DNA can be obtained by standard procedures, 19growing the bacteria at 30°. Compared to several other routinely used plasmids the yield of DNA is relatively low. Higher yields are obtained with the pEX-derived vector pUEX 2° which can be grown in bacterial strains other than E. coli pop 2136 and which is available commercially (Amersham International, Ltd., Amersham, England).

Preparation of Fusion Proteins Three milliliters of LB medium containing 100/zg ampicillin (LB-amp) is inoculated with a bacterial colony and grown overnight at 30°. A 2.5-ml portion of this culture is used to inoculate 250 ml of L B - a m p for a maximal preparation of fusion protein (Step B). The bacteria are grown under vigorous shaking to an OD600 of 0.4. An equal volume of L B - a m p heated to 54° is added, and the bacteria are shaken for 2 hr at 42 ° in a water bath. The bacteria are centrifuged for 10 min at 3000 g and 4°C (Step D). The pellets are resuspended in 10 ml of 15% saccharose, 50 mM Tris-HC1 (pH 8.0), 50 mM EDTA and transferred to Corex tubes. Ten milligrams of lyzozyme is added, and the tubes are incubated with occasional shaking on ice for 40 min, after which 14 ml of 0.2% Triton X-100 is added. After incubation on ice for 5 min the cells are strongly sonified, transferred to an ultracentrifuge tube, and centrifuged at 35,000 g for 15 min at 4 °. The resulting pellet contains the fusion protein, which is resuspended in phosphate-buffered saline (PBS; 10 mM potassium phosphate, pH 7.5). For minipreparations of fusion proteins the procedure is modified as follows. In Step B, 25/.d of the bacterial culture grown overnight is used to inoculate 2.5 ml of LB-amp. After Step D the bacteria are resuspended in 100/xl PBS. This bacterial suspension can be analyzed by SDS-PAGE.

Analysis and Purification of Fusion Proteins Fusion proteins are analyzed by SDS-PAGE. Up to 1 mg of fusion protein can be purified by SDS-PAGE with a separating gel containing 6% acrylamide and 0.15% (w/v) bisacrylamide. After the electrophoresis 19 j. Sambrook, E. F. Frisch, and T. Maniatis, "Molecular Cloning: A Laboratory Manual," 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989. 20 G. M. Bressan and K. K. Stanley, Nucleic Acids Res. 15, 10056 (1987).

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is completed, the gel is stained for 10 min with Coomassie blue in 10% acetic acid, destained, and the gel piece containing the fusion protein excised. In principle, the fusion protein should constitute the major cellular protein. The excised gel piece is placed into a dialysis bag which had been previously boiled in 50 mM EDTA, pH 8, and rinsed with water. The dialysis bag is filled with elution buffer (25 mM Tris base, 190 mM glycine, and 0.1% SDS, final pH 8.3). The closed dialysis bag is placed into a small electrophoresis chamber, and the protein is eluted at 5 V/cm for 1 hr. Then the electrical field is reversed. After 2 min the protein solution is removed.

Immunization Procedures Rabbits are immunized subcutaneously with 500 /zg of antigen in Freund's complete adjuvant followed by two immunizations with 200/zg of antigen in Freund's incomplete adjuvant. Each of the immunizations takes place 4 weeks apart. The antisera are tested by immunoblotting against microsomal protein and against the antigen. When the sera are positive, the animal is anesthetized and bled out. Repeated immunizations are not advisable, because the antisera specificity changes. It is important to note that the specificity of some sera might be dependent on the conditions used for the incubation of the immunoblot with the first antibody.

Purification of Antibodies Antibodies can be purified by affinity chromatography on 2 columns containing the fusion protein used as an antigen linked to Sepharose following passage of the antiserum over a column containing the cro-lacZ protein linked to Sepharose. Because these proteins are relatively insoluble a special procedure has to be used for coupling to Sepharose: 7 mg of the ligand is dissolved in coupling buffer (0.1 MNaHCO3, pH 8.3, 0.5 M NaCI, and 7 M urea). The protein solution is incubated overnight with 4 ml of swollen cyanogen bromide-activated Sepharose. Under these conditions urea cannot bind to the resin. The affinity material is washed with the coupling buffer. The resin is further blocked with 0.2 M glycine, pH 8, and washed with 0.1 M sodium acetate, pH 4, 0.5 M NaCI and with 0.1 M Tris-Cl, pH 8, containing 0.5 M NaCI. For affinity chromatography the column is equilibrated with phosphatebuffered saline. Five milliliters of antiserum is first passed through a column containing the cro-lacZ protein. The flow through is applied onto a column containing the relevant antigen. Antipeptide antibodies are eluted with 0.2 M glycine, pH 2.2, into a buffer containing 1 M Tris-C1, pH 9. The antibodies are stored at - 2 0 ° and are used for immunodetection.

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FIG. 2. Restriction map of the P450IIB 1 and P450IIB2 cDNAs. Shown are the restriction sites that were used to subclone the various restriction fragments (shown as black or hatched boxes) into the expression vectors. The restriction fragments from the IIB1 eDNA were designated A, B, CI, D, and E and the one from the IIB2 eDNA as C2. Also shown are the amino acid sequences coded for by the subcloned restriction fragments in the fusion proteins. D2 and E2 are restriction fragments in the IIB2 eDNA which correspond to D and E. These fragments were not subcioned into the expression vectors.

Results Several restriction fragments of the P450IIB1 and P450IIB2 cDNAs were subcloned into the pEX vectors. Figure 2 shows the location of these restriction fragments on the P450 cDNAs and the amino acid sequences encoded by them on the mature proteins. The length of the peptides varied from 18 to 33 amino acids. With the exception of the fusion protein containing the amino terminus of P450IIB 1, all other fusion proteins elicted antibodies which were reactive with P450IIB 1 and/or P450IIB2. It is interesting to note that others 7 also did not succeed in generating antibodies directed against the amino-terminal portion of P450IIB 1 by using synthetic peptides coupled to a carrier protein. Figure 3 shows the reactivity of the various anti-P450 fusion protein antibodies with microsomal protein and with purified P450IIB1 and P450IIB2 on immunoblots. Antibodies for these experiments had been purified by affinity chromatography on columns containing the cro-lacZ protein followed by affinity chromatography on columns containing the

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IMMUNOCHEMICAL METHODS

a

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1M

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FIG.3. Westernblotsof purifiedP450IIB1(lanes 1), P450IIB2(lanes2), and ofmicrosomal proteinfromphenobarbital-treatedrats (lanesM). One-halfmicrogramofthe purifiedproteins and 50/zg of microsomalprotein were applied to the gel. The immunoblotswere incubated with the followingantibodies:(a) polyclonalpurifiedanti-P450IIB1; (b-f) antipeptideB, C1, D, E, and C2 antibodies, respectively. relevant antigen. For most purposes, however, it is sufficient to purify the antisera on a column containing the cro-lacZ protein. Antibodies directed against the constant region of P450IIB 1 and P450IIB2, namely, antipeptide B and E antibodies, recognize both cytochromes P450 as well as a third protein (termed X) with a higher apparent molecular weight. Antibodies directed against the hypervariable region of P450IIB2, namely, antipeptide C2 antibodies, recognize only this P450 and protein X but do not recognize P450IIB 1. However, antibodies directed against peptide C I which covers a region on P450IIB 1 which corresponds to the region covered by peptide C2 on P450IIB2 react with P450IIB 1 and P450IIB2 even though both P450s differ in this region by 4 out of 33 amino acids. Antipeptide D antibodies which were directed against a region where P450IIB 1 and P450IIB2 differ by only 2 of 19 amino acids, however, preferentially recognize P450IIB 1. We have also tested the reactivity of the antifusion protein antibodies toward nondenatured in vitro synthesized P450IIB1 and P450IIB2. s Only antipeptide E antibodies were reactive toward nondenatured P450IIBI and P450IIB2. Conclusions In most cases efficient production of antibodies directed against short P450 sequences on fusion proteins can be achieved. Unlike site-directed P450 antibodies which are directed against synthetic peptides and which

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mainly recognize native cytochromes P450,7 the antibodies directed against P450 fusion proteins recognize mainly denatured cytochromes P450. We were also successful in generating antibodies against P450 fusion proteins containing almost full-length P450IIC6 or P450IIC7 fusion protein. The antibodies recognized these proteins in microsomes. Thus, fusion proteins can be used to generate antibodies against a cytochrome P450 which is not available in a purified form in the laboratory. Acknowledgments We thank Mrs. I. Bfhm and Mrs. H. Steedfor typingthe manuscript.This work was supported by the DeutscheForschungsgemeinschaft(SFB 302).

[20] I m m u n o i s o l a t i o n of H u m a n M i c r o s o m a l C y t o c h r o m e s P 4 5 0 Using A u t o a n t i b o d i e s

By

ULRICH M.

ZANGER

Introduction Human cytochrome P450 isozymes of three P450 subfamilies have recently been shown to be specifically recognized by certain types of circulating autoantibodies, which occur in patients with either drug-induced or idiopathic chronic active forms of hepatitis (Table I; see also [21], this volume). All these P450 isozymes are mainly expressed in the liver, they metabolize xenobiotic compounds, and their catalytic function is potently inhibited by the respective autoantibodies. Besides the interest evoked by these autoantibodies in the mechanisms leading to autoimmunity and drug-induced hepatitis, they have proved to be highly valuable immunological tools of amazing specificity and potency. The protocols presented in this chapter describe the use of autoantisera to immunoisolate P450 proteins from solubilized microsomes. They may of course be applicable to other types of autoantibodies as well as to antibodies raised in rabbits. A microscale procedure is presented, which allows one to detect and semiquantitatively isolate microsomal antigens. By immunopurification on a larger scale, sufficient amounts of highly pure antigen can be obtained for further protein chemical analysis and for the production of antibodies. These methods have been used with the socalled anti-LKM (anti-liver/kidney microsome) antibodies type 1 and 2 to detect and identify their autoantigens, the liver microsomal P450IID6 and METHODS IN ENZYMOLOGY, VOL. 206

Copyright© 1991by AcademicPress, Inc. All rights of reproduction in any form reserved.

Production of site-specific P450 antibodies using recombinant fusion proteins as antigens.

[19] PRODUCTION OF SITE-SPECIFIC P450 ANTIBODIES 193 [19] P r o d u c t i o n o f Site-Specific P 4 5 0 A n t i b o d i e s Using R e c o m b i n a...
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