His-Tagged Human Peptide Transporter hPEPT1 in Yeast for Protein Purification and Functional Analysis

Protein Expression and Purification 22, 436–442 (2001) doi:10.1006/prep.2001.1455, available online at http://www.idealibrary.com on

Expression of the myc/His-Tagged Human Peptide Transporter hPEPT1 in Yeast for Protein Purification and Functional Analysis Stephan Theis, Frank Do¨ring, and Hannelore Daniel1 Institute of Nutritional Sciences, Molecular Nutrition Unit, Technical University of Munich, Hochfeldweg 2, D-85350 Freising-Weihenstephan, Germany

Received January 29, 2001, and in revised form April 1, 2001; published online July 17, 2001

The human intestinal peptide transporter hPEPT1 has been expressed in the yeast Pichia pastoris using the promoter of the glyceraldehyde-3-phosphate-dehydrogenase gene. A myc-epitope fused to a polyhistidine-tag was introduced at the C-terminus of hPEPT1 for ease of detection and purification. Yeast cells transformed with tagged hPEPT1 exhibited 30-fold increased dipeptide uptake compared to control cells with a substrate specificity and pH dependence similar to the native transporter. The tagged hPEPT1 protein was detected in crude membrane fractions of Pichia cells with an apparent molecular mass of 66 kDa and an expression level of approximately 64 pmol/mg membrane protein. These studies demonstrate that tagged hPEPT1 can be expressed functionally in P. pastoris with unaltered phenotypical characteristics allowing the yeast cells to be used for functional analysis such as screening for compounds utilizing the peptide transporter for absorption in the human intestine. Moreover, recombinant hPEPT1 can now easily be detected for further purification purposes using immobilized metal-affinity chromatography. 䉷 2001 Academic Press Key Words: human intestinal peptide transporter; Pichia pastoris; myc-epitope; polyhistidine-tag.

The human intestinal peptide transporter hPEPT12 is responsible for the absorption of di- and tripeptides 1 To whom correspondence should be addressed. Fax: ⫹49 8161 713 999. E-mail: [email protected]. 2 Abbreviations used: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; hPEPT1, human intestinal peptide transporter; T.U., turbidity of the cell suspension measured at 600 nm (1T.U. equals approximately 5 ⫻ 107 cells); D-Phe-Ala, D-phenylalanyl-L-alanine; IgG, immunoglobulin G; PPB, potassium phosphate buffer; PMSF, phenyl-

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in epithelial cells of the small intestine (1–3). One of the unique features of the intestinal peptide transporter is its capability to transport all possible di- and tripeptides as well as numerous peptidomimetic drugs. In addition, it is a high-capacity transporter making it an ideal target for efficient drug delivery (4–8). Although there has been some progress in understanding the basic characteristics of substrate recognition by hPEPT1, so far no predictive model allowing a rational design of drugs utilizing this transporter has been proposed. Screening for pharmacologically interesting peptidomimetics that target hPEPT1 for drug delivery requires a standardized assay system with a highthroughput screening potential. Most of the heterologous expression systems used for analysis of cloned transporter proteins are based on transfected mammalian cell lines or Xenopus laevis oocytes but in all cases they do not allow such high throughput applications and are also very expensive. Yeast cells that can be grown in large quantities and in cheap media however could provide such a platform for drug screening. Moreover, transgenic yeast such as Pichia pastoris can be grown to high cell densities that provide a good starting point for transporter protein production and purification. Although there have been different attempts to purify and characterize the peptide transporters on the protein level (9, 10) these studies obtained only trace quantities of proteins with functions mostly not comparable with the characteristics of the cloned peptide transporters of the different mammalian species (3,

methylsulfonyl fluoride; PI, protease-inhibitor cocktail; BSA, bovine serum albumin; PVDF, polyvinylidene difluoride. 1046-5928/01 $35.00 Copyright 䉷 2001 by Academic Press All rights of reproduction in any form reserved.

TAGGED HUMAN PEPTIDE TRANSPORTER IN YEAST

11–13). This may mainly be due to the very low abundance of the proteins in their native tissues when these sources are used but other due to limitations such as very heterogeneous starting material comprising different cell types and a large number of interfering proteins. For this reason a suitable expression system with the ability to produce large amounts of the desired protein with conserved functional properties is in demand. We have previously shown (14, 15) that P. pastoris can be used to express the mammalian peptide transporters. In this study we have extended this preliminary study by using expression vectors including tags that have been added to the carboxyterminus of human PEPT1 for allowing its purification from yeast cell membranes. One of the selection criteria for the heterologously expressed tagged hPEPT1 in yeast was the maintenance of all functional characteristics. The advantages of the system described here is that it provides large amounts of yeast cells for utilization in screening for potential substrates based on the preserved protein function in yeast and at the same time allowing the first steps of protein isolation with the ease of the tags incorporated. MATERIALS AND METHODS Materials. D-Phenylalanyl-L-alanine (D-Phe-Ala) was purchased from Bachem (Heidelberg, Germany). Custom synthesized D-[3H]Phe-Ala with a specific radioactivity of 40 Ci/mmol was obtained from Biotrend (Cologne, Germany). Cyclacillin was a generous gift from Dr. M. Brandsch (Bio-Zentrum, Halle, Germany). Amino acids, peptides, peptidomimetics, high-molecular-weight-range marker, anti-mouse IgG (peroxidase conjugate), 3-amino-9-ethyl-carbazole, and protease-inhibitor cocktail were obtained from Sigma (Deisenhofen, Germany). Positope protein, anti-myc antibody, and Zeocin were obtained from Invitrogen (San Diego, CA). Restriction enzymes and DNA markers were purchased from New England Biolabs (Schwalbach, Germany). DNA polymerase (elongase amplification system) was obtained from Life Technologies (Karlsruhe, Germany). All other chemicals were purchased from Roth (Karlsruhe, Germany). Strains and transformations. Escherichia coli strain XL1-Blue (Stratagene, Heidelberg, Germany) was used for propagation and transformation of the different expression plasmids (16). P. pastoris strain GS115 deficient in histidinol dehydrogenase activity (Invitrogen) was used for all expression studies. Transformations of yeast cells were performed according to the lithium chloride method as described in the manual Version E of the Pichia expression kit (Invitrogen). Construction of vectors for expression of hPEPT1. For myc- and His-tagging of hPEPT1 the plasmid pGAPZB-hPEPT1myc-His was constructed. The whole

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open reading frame of the hPEPT1 cDNA was first amplified from the plasmid pBluescript SK-hPEPT1 by PCR. This fragment was constructed to contain a XbaI restriction site in the native stop codon of the hPEPT1 cDNA. The fragment was then inserted into the AvrII/ XbaI sites of the vector pCRII (Invitrogen). From the resulting plasmid the hPEPT1 fragment was introduced into the SfuI/XbaI sites of pGAPZB (Invitrogen) leading to in frame fusion of the hPEPT1-cDNA with C-terminal myc-epitope and polyhistidine (His)-tag of the pGAPZB plasmid. Constructed plasmid was verified by sequence analysis prior to functional studies. Transformation of yeast cells and selection of multicopy integrands. P. pastoris cells were transformed with AvrII-linearized plasmid pGAPZB– hPEPT1myc-His or pGAPZB (control). Transformants were selected by growth on YPD (1% yeast extract, 2% peptone, 2% glucose) agar plates supplemented with Zeocin (100 ␮g/ml). The identification of multicopy clones containing several copies of pGAPZB or pGAPZB–hPEPT1myc-His constructs in the genome is based on the selection of Zeocin hyperresistant transformants (up to 1.2 mg/ml Zeocin) as described (15). Transport studies in the yeast P. pastoris expressing tagged hPEPT1. For uptake measurements, cells of the clones pGAPZB–hPEPT1myc-His and pGAPZB (control) were grown to a turbidity of 4–6 T.U./ml in YPD medium. Cells were pelleted at 3000g for 10 min, washed twice with 100 mM potassium phosphate buffer (PPB, pH 6.5) and resuspended to 1 T.U. ⫻ 20 ␮l⫺1 PPB. Uptake measurements were performed at 22–24⬚C by using a rapid filtration technique either using single tubes and single filter plates (Schleicher & Schuell, ME25 type, 0.45 ␮M pore size) (14) or on 96-well filter plates (HATF type, 0.45 ␮M pore size; Millipore, Eschborn, Germany). In brief, uptake was initiated by mixing 20 ␮l of cell suspension with 30 ␮l of PPB containing 0.1 ␮Ci D-[3H]Phe-Ala either with or without competitors (final concentration 0.001–10 mM). After 20 min of incubation, uptake was terminated by addition of 200 ␮l of ice cold PPB followed by filtration. The filters were washed four more times with 200 ␮l of PPB, in the case of the 96-well filter plates removed from the plate with a punch and transferred into vials. Radioactivity associated with the filter was counted by liquid scintillation counting (Beckman LS6500, Fullerton, CA). Membrane preparations. Cells cultured in YPD medium were harvested at a turbidity of 4–6 T.U./ml by centrifugation at 3000g and 4⬚C for 10 min, washed once with ice-cold breaking buffer (50 mM sodium phosphate, pH 7.4, 1 mM EDTA, 5% glycerol), and resuspended to 100 T.U./ml breaking buffer supplemented with 1 mM PMSF and 2 ␮l/ml protease-inhibitor cocktail (PI). An equal volume of acid-washed glass beads (0.5 mm diameter) was added to the suspension, and

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cells were disrupted by vigorous vortexing eight times for 30 s, with intervening 30-s incubations on ice. Unbroken cells were removed by centrifugation at 2000g and 4⬚C for 10 min. Membranes were pelleted at 100,000g and 4⬚C for 30 min and resuspended in membrane suspension buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA) including 1 mM PMSF and 1 ␮l/ml PI. Aliquots were snap-frozen and stored at ⫺80⬚C. The protein concentration of the membrane preparation was determined using the Roti-Quant method (Roth, Karlsruhe, Germany) with BSA as standard. SDS–PAGE and immunoblotting. Yeast membrane proteins or the control protein (positope protein, Invitrogen) was separated by SDS–PAGE (10% gel) using a Mini-Protean II electrophoresis cell (Bio-Rad, Munich, Germany) and electrophoretically transferred to

PVDF membrane (NEN, Boston, MA). The membrane was blocked for 1 h at room temperature with 4% nonfat powdered milk in Buffer A (137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4). For immunodetection of myc-tagged protein, the monoclonal mouse anti-myc antibody was used at a final concentration of 0.9 ␮g/ml. Incubation with the primary antibody was performed overnight at 4⬚C and with the secondary antibody (anti-mouse IgG, peroxidase conjugate, diluted 1:2000) 1 h at room temperature. For visualization of the myc-tagged proteins the blots were developed in 10 ml 50 mM Na-acetate buffer, pH 5.0 containing 4 mg 3-amino-9-ethylcarbazole and 5 ␮l 30% H2O2. For quantification of the myc-tagged protein, blots were analyzed by densitometry using the SigmaGel analysis software. Calculations and statistics. All calculations (linear

FIG. 1. (A) Uptake of D-Phe-Ala into P. pastoris cells transformed with the tagged hPEPT1 construct. P. pastoris cells were transformed with the plasmid pGAPZB–hPEPT1myc-His. The empty vector pGAPZB served as control. For both plasmids, 10 to 20 transformants were tested for uptake of 5 ␮M D-[3H]Phe-Ala (2 ␮Ci/ml) for 20 min of incubation at a pH value of 6.5 (single test tube assay). The boxes shown extend from the 25th percentile to the 75th percentile, with a horizontal line at the median (50th percentile) of uptake rates. Whiskers extend down to the lowest value and up to the highest values measured. PGAP, promoter of P. pastoris GAPDH gene; Zeo, Zeocin resistance gene; myc, His, coding regions for the myc -and His-tag. (B) Uptake of D-Phe-Ala as a function of cell density. Uptake of 5 ␮M D-[3H]PheAla (2 ␮Ci/ml) into P. pastoris cells expressing tagged hPEPT1 or control cells was measured for 20 min of incubation at different optical densities of the yeast cultures. (C) Uptake of D-Phe-Ala as a function of extracellular pH. Influx of 5 ␮M D-[3H]Phe-Ala (2 ␮Ci/ml) into P. pastoris cells expressing tagged hPEPT1 or control cells was measured for 20 min of incubation in phosphate buffers of pH 5.0 to 8.0. (D) D-Phe-Ala uptake as function of substrate concentration. Uptake of D-[3H]Phe-Ala (2 ␮Ci/ml) into the P. pastoris cells expressing tagged hPEPT1 was measured for 20 min at pH 6.5 in the presence of increasing concentrations of unlabeled substrate (0.0001 to 25 mM). The resulting net transport rates were transformed according to Eadie–Hofstee (inset) and kinetic constants were derived by linear regression analysis by the least-squares method (r 2 ⱖ 0.95).

TAGGED HUMAN PEPTIDE TRANSPORTER IN YEAST

as well as nonlinear regression analysis) were performed by using Prism (GraphPAD, Los Angeles, CA). For each variable at least two independent experiments with three replicates were carried out. Data are given as the mean ⫾ SEM. RESULTS AND DISCUSSION Expression of a Tagged hPEPT1 in the Yeast P. pastoris The cDNA coding for the human intestinal peptide transporter hPEPT1 was cloned into an E. coli/P. pastoris shuttle vector to express the carrier protein constitutively in the yeast P. pastoris. A myc-tag fused to a polyhistidine-tag was introduced at the C-terminus of hPEPT1 to facilitate immunological detection and for allowing purification. The plasmid construct (Fig. 1A) was transformed into the P. pastoris wild-type strain GS115. The empty vector served as a control. As shown in Fig. 1A, the functional expression levels of tagged hPEPT1 (hPEPT1myc-His) were different for each of the various transformants tested. However, by selecting for clones with multiple integrations of the hPEPT1myc-His construct we obtained 5- to 20-fold higher uptake rates than in the nonselected clones. One clone containing

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the hPEPT1myc-His construct showed an up to 30-fold higher uptake rate of D-[3H]Phe-Ala than the isogenic control strain. Since the level of expression by the GAPDH promoter depends on the cell density of the yeast culture, we optimized our harvesting and uptake conditions. As shown in Fig. 1B, the highest transport activity of tagged hPEPT1 was obtained at higher cell densities. Therefore, the selected hPEPT1myc-His strain and the optimized culture conditions were used for further functional characterization and detection of the tagged hPEPT1. Transport Characteristics of Tagged hPEPT1 The rationale for incorporation of the tags into the carboxyterminus of hPEPT1 was based on previous studies with chimeric peptide transporters that suggested that fusion of additional amino acid residues onto the C-terminus of the protein might not impair its function (17). This was confirmed by determining pH dependency, substrate affinity, and substrate specificity of tagged hPEPT1 after expression in P. pastoris. As shown in Fig. 1C, tagged hPEPT1-mediated the uptake of D-[3H]Phe-Ala with a strong dependence on extracellular pH and a pH optimum at 6.5. A very similar pH

A

B

C

D

FIG. 2. Inhibition of D-Phe-Ala uptake by different compounds in tagged hPEPT1 expressing P. pastoris cells. Uptake of D-[3H]Phe-Ala (2 ␮Ci/ml) into P. pastoris cells expressing tagged hPEPT1 was measured for 20 min of incubation at pH 6.5 on 96-well filter plates in absence or presence of increasing concentrations (0.001–10 mM) of competitors. Data are presented as the mean ⫾ SEM for the residual uptake of D-[3H]Phe-Ala in the presence of inhibitors (percentage of control uptake).

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dependence of transport function of PEPT1 has been obtained in mammalian cell systems or in X. laevis oocytes expressing the protein (3). Studies on the D[3H]Phe-Ala uptake into transgenic yeast cells as a function of increasing substrate concentrations revealed the Michaelis–Menten type saturation kinetics shown in Fig. 1D. The affinity of D-Phe-Ala for transport by tagged hPEPT1 revealed an approximately K0.5 of 1.18 ⫾ 0.24 mM which is in excellent agreement with transport data obtained with yeast cells transformed with the native hPEPT1 or by using other expression systems (3, 15). To extend the phenotypical characterization of tagged hPEPT1 we also determined its substrate specificity. Selective interaction of peptides with the substrate binding site of tagged hPEPT1 was established by competition studies. As shown in Fig. 2, the uptake of D-[3H]Phe-Ala was inhibited by neutral as well as charged di- and tripeptides in a dose-dependent manner. In contrast, neither larger peptides nor free amino acids interacted with hPEPT1myc-His. Incorporation of a D-isomer into the N- and/or C-terminal position of the dipeptide Ala-Ala reduced the affinity to hPEPT1 in the following order: L-Ala-L-Ala ⬎ D-Ala-L-Ala ⬎ LAla-D-Ala ⬎ D-Ala-D-Ala, showing the enantioselectivity of substrate recognition by tagged hPEPT1. We recently demonstrated that the nonpeptide and precursor of porphyrin synthesis ␦-aminolevulinic acid is a substrate of rabbit peptide transporters (18). Here we show that ␦-aminolevulinic acid also similarly interacts with the human intestinal peptide transporter. Taken together, the obtained IC50 values for the different substrates (Table 1) establish that the tagged hPEPT1 retains its substrate affinity and specificity and that the myc-/polyhistidine-tag introduced at the C-terminus of the hPEPT1 protein obviously not impairs its basic functional characteristics. Interaction of ␤-Lactam Antibiotics with the Tagged hPEPT1 As one of the applications of the P. pastoris system expressing the tagged hPEPT1 with all its functional features we determined the affinities of selected cephalosporin and penicillin antibiotics with hPEPT1myc-His in yeast based on a 96-well microplate assay. As PEPT1 is the main route by which orally active ␤-lactam antibiotics are transported into the epithelial cells of the intestine our transgenic yeast cells could be a useful tool for identifying compounds interacting with hPEPT1. We therefore selected 13 different ␤-lactam antibiotics covering a wide range of structures and known to display quite different absorption rates in vivo in humans. Figures 2C and 2D show the competition curves and Table 1 shows the corresponding IC50 values. Compounds such as cefadroxil, cefaclor, cephradine, cephalexin, cefixime, or cyclacillin—all known to be orally

TABLE 1 Affinity of Selected Compounds for Inhibition of D-PheAla Influx into hPEPT1myc-His-Expressing Yeast Cells Compounds

IC50 values (mM) ⬎25 0.1 ⫾ 0.01 0.2 ⫾ 0.03 ⬎10

L-Ala L-Ala-L-Ala L-Ala-L-Ala-L-Ala L-Ala-L-Ala-L-Ala-L-Ala

D-Ala-D-Ala

0.8 ⫾ 0.1 6.1 ⫾ 0.2 ⬎10

Gly-Lys Gly-Asp Ala-His

0.3 ⫾ 0.03 1.0 ⫾ 0.3 0.3 ⫾ 0.4

␦-Aminolevulinic acid Cephalosporins Cefadroxil Cephaloglycin Cefaclor Cephradine Cefixime Cefamandole Cephalexin Cephalothin Cefuroxime Ceftriaxon Penicillins Cyclacillin Benzylpenicillin Ampicillin

0.5 ⫾ 0.1

D-Ala-L-Ala L-Ala-D-Ala

1.4 1.5 1.6 6.0 6.3 8.0 8.2 11.2 11.2 25.3

⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾

0.1 0.3 0.2 1.4 1.2 2.6 1.8 3.4 0.2 2.0

0.6 ⫾ 0.1 21.2 ⫾ 3.4 25.6 ⫾ 1.3

Note. IC50 values were derived by nonlinear regression analysis from inhibition plots shown in Fig. 2.

available—displayed inhibition constants comparable to those of normal di- or tripeptides in the range from 0.6 to 8 mM. In contrast, the cephalosporins such as ceftriaxon, cephalothin, or cefuroxime and benzylpenicillin, considered to be nonorally active and usually administered parenterally, showed inhibition constants of higher than 10 mM. Comparing these affinities from transgenic yeast cells with recently published data obtained in Caco-2 cells expressing wild-type hPEPT1 (19) reveals that the yeast cells expressing tagged hPEPT1 are suitable for predicting the interaction of compounds with the intestinal carrier in vivo. The simplicity and ease of handling in combination with the developed uptake assay on 96-well microplates makes our system useful for high-throughput screening applications for drugs which utilize the peptide transporter for absorption in the human intestine. Detection and Quantification of the Tagged hPEPT1 Protein Expression of the tagged hPEPT1 in yeast cells was further analyzed by immunoblotting. Membrane protein fractions isolated from transgenic yeast cells expressing the tagged hPEPT1 and separated by SDS–

TAGGED HUMAN PEPTIDE TRANSPORTER IN YEAST

PAGE revealed the presence of an intense signal in the Western blot by using the anti-myc antibody (Fig. 3A, lanes 1–4). In contrast, no signal was obtained in membranes prepared from the control strain (Fig. 3, lane 5). The apparent molecular mass of the tagged hPEPT1 protein was around 66 kDa. Considering the molecular mass of the C-terminal tag with 2.5 kDa, hPEPT1 shows a relative molecular mass of about 63 kDa. Thus, the P. pastoris-expressed hPEPT1 protein migrates slightly faster on SDS–PAGE than expected from its amino acid sequence with a predicted molecular size of 78.8 kDa (3). However, in vitro translation of the cRNA coding for the rabbit transporter isoform with an almost identical predicted molecular weight also resulted in only a 60kDa signal on SDS–PAGE for the nonglycosylated protein (12). To determine the amount of tagged hPEPT1 protein

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produced in transgenic yeast cells we used the recombinant 53-kDa myc-tagged protein positope for standardization. As shown in Fig. 3B, we separated different amounts of the control protein by SDS–PAGE, blotted the proteins electrophoretically, and detected the bands with the anti-myc antibody. By linear regression analysis of the densities of the signals obtained for different amounts of loaded positope protein we obtained the standard curve shown in Fig. 3B. This allowed the quantification of tagged hPEPT1 protein in crude membrane fractions from the transgenic yeast cells. The optical densities of the resulting signals were determined to correspond to 142, 403, 599, and 705 ng tagged hPEPT1 protein that accounted for 5–7 ng/␮g total membrane protein corresponding to 0.5 to 0.7% of total membrane protein isolated. Based on the amino acid sequence of hPEPT1 and the calculated relative molecular mass of 78,800 a total of about 64 pmol hPEPT1 protein was found per milligram of membrane protein. This is in the range of yield obtained with other membrane proteins expressed in P. pastoris such as the bovine opsin (20) or selected G-protein-coupled receptors (21, 22). Considering that from 1 T.U. yeast cells about 10 ␮g of membrane protein could be prepared, 1 liter of transgenic yeast cells grown to 4 T.U./ml can yield about 0.2 mg of hPEPT1 protein which might prove to be sufficient for further protein purification procedures. CONCLUSIONS We here show that P. pastoris allows the tagged human peptide transporter hPEPT1 to be expressed with fully preserved functional characteristics. The myc-epitope introduced enables with ease of detection the further optimization of protein production and the fused polyhistidine-tag will provide a tool for further protein purification attempts. ACKNOWLEDGMENTS This work was supported by the Deutsche Forschungsgemeinschaft (Da 190/5-1) to H.D. We thank M. A. Hediger for providing the cDNA of hPEPT1 and Dr. Matthias Brandsch for providing Cyclacillin. REFERENCES

FIG. 3. Immunoblot analysis of tagged hPEPT1 expressed in P. pastoris. Yeast membrane proteins and positope protein separated by SDS–PAGE were blotted onto PVDF membrane and probed with monoclonal anti-myc antibody. The antibody recognizes the myc-epitope C-terminally fused to the hPEPT1 protein or being part of the positope protein. (A) Lanes 1–4, membrane protein of hPEPT1 expressing yeast cells; lane 1, 25 ␮g; lane 2, 50 ␮g; lane 3, 75 ␮g; lane 4, 100 ␮g. Lane 5, 100-␮g membrane protein of the control strain (con). (B) Linear regression of optical densities of the signals obtained by immunoblotting (inset) increasing amounts of positope protein.

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