108
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[11]
sis 32 o r chimeric protein constructs) These types of experiments can give insight into the structural basis of P450 substrate specificity, but other expression systems such as yeast, baculovirus, or Escherichia coli may be better suited to obtain the quantity of P450 required for spectral analysis.
Acknowledgments This research was supported by a grant from the National Institutes of Health (GM 37429). B. J. C. is supported in part by a predoctoral fellowship from Merck, Sharp and Dohme Research Laboratories, Rahway, New Jersey. 32 R. L. Lindberg and M. Negishi, Nature (London) 339, 632 (1989).
[11] E x p r e s s i o n o f M a m m a l i a n P 4 5 0 s in Escherichia coli
By TODD D. PORTER and JANE R. LARSON Introduction
Escherichia coliis a desirable organism for the heterologous expression of proteins owing to its ease of manipulation, the availability of a variety of cloning and expression vectors, well-understood genetics, and the low cost of culture. The expression in bacteria of membrane-bound proteins, however, has proved less efficient than that of cytosolic proteins, presumably because of the lack of intracellular membranes into which these hydrophobic proteins can sequester. Whereas an expressed cytosolic protein may constitute up to 30% of the total cellular protein, expressed membrane-bound proteins typically constitute less then 1% of the total cellular protein. Nonetheless, the segregation of these proteins into the inner membrane of the cell allows subcellular fractionation to serve as a convenient and efficient first step in the isolation of the heterologously expressed protein.
Expression Vectors, Culture, and Induction
Vectors We have examined several types of vectors for expression of P450 in E. coli; critical factors for optimal expression are the strength of the promoter and the number of copies of the plasmid in the cell. A strong METHODSIN ENZYMOLOGY,VOL. 206
Copyright© 1991by AcademicPress,Inc. All rightsof reproductionin any formreserved.
[11]
EXPRESSIONOF MAMMALIANP450s IN E. coli
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promoter, such as the t r p - l a c fusion promoter trc, 1 and a high copy number, as obtained with the origin of replication from the pUC series of plasmids, 2 yield the highest levels of expression. A third factor which appears to be essential to the stable maintenance of the clone is the use of a regulatable promoter, such that expression of the heterologous protein is constitutively suppressed but can be induced when appropriate. We have used both the temperature-sensitive expression vector pCQV2, 3 which utilizes the h rightward promoter and the temperaturesensitive ci857 repressor, and the chemically inducible expression vector pKK233-2, 4 which utilizes the isopropylthiogalactoside-inducible (IPTG) trc promoter. Although we find the trc promoter to be 3-fold stronger than the h promoter, the pCQV2 system offers the advantage of being less expensive to use, as induction of expression is achieved without the use of the relatively expensive chemical, IPTG. This expense becomes relevant when large-scale cultures (200 liters) are anticipated. An additional advantage of the pCQV2 vector is strain flexibility: the ci857 repressor is encoded on the plasmid, and thus the plasmid is not restricted to h lysogens. In this context, it should be noted that the level of expression of a related enzyme, rat NADPH-cytochrome P-450 reductase, has been shown to differ among various strains of E . c o l i : A disadvantage of the pCQV2 vector is the need to grow the cells at 30° to suppress heterologous protein expression while bringing the cells to late log-phase density, and the subsequent need to rapidly shift the temperature to 37° to induce expression. Whereas one-half volume of prewarmed media (at 42°) can be added to standard cultures to quickly raise the temperature, this is not feasible with large cultures. Our experience indicates, however, that P450 expression is not immediately toxic to the cells, and thus a more gradual increase in temperature, as achieved in large-scale fermentors, is not likely to be detrimental. The trc promoter of pKK233-2 offers a higher level of expression than the h promoter, but it suffers the disadvantage of being restricted to strains which contain the l a c l q gene. These strains (such as JM 1052) constitutively overexpress the l a c I repressor, and thus suppress transcription from the trc promoter in the absence of inducer. Induction of expression requires the addition of IPTG to at least 0.5 mM. The two cytochrome P450 expression vectors we have constructed are 1 j. Brosius, M. Ertte, and J. Storella, J. Biol. Chem. 260, 3539 (1985). 2 C. Yanish-Perron, J. Vieira, and J. Messing, Gene 33, 103 (1985). 3 C. Queen, J. Mol. Appl. Gen, 2~ 1 (1983). 4 E. Amann and J. Brosius, Gene 40, 183 (1985). 5 T. D. Porter, T. E. Wilson, and C. B. Kasper, Arch. Biochem. Biophys. 254, 353 (1987).
110
HETEROLOGOUSEXPRESSION
[11]
Eoo RI
Nco I Eco RI
/¢
°''
pCO3a ! Ind III
Pvu I
~('Xho I/Sell I) Hind III FIG. 1. Cytochrome P450 expression plasmids.
shown in Fig. 1. Both plasmids contain a full-length cytochrome P450 2El c D N A 6'7 encoding the rabbit alcohol-inducible form of the cytochrome
(P450 3a). s The presence of an N c o I restriction site at the translation initiation codon (CCATGG) allowed us to conveniently insert the 2El cDNA into an appropriate cloning site in each vector: N c o I for pKK233-2 and B a m H I for pCQV2. In the latter construct, the BarnHI and N c o I ends of the DNA were made flush with mung bean nuclease prior to ligation. Both vectors contain a ribosome binding site upstream of the cloning site. pCQ3a is derived from pCQV2) and thus expression is induced by a temperature shift to inactivate the ci857 repressor. By replacing the pBR322 origin of replication with that from a pUC-derived plasmid, we obtained an 8-fold greater level of expressed protein, as determined by immunoblotting, which we attribute to the 3- to 5-fold higher copy number for plasmids containing this origin. 9 pKK3a is derived from pKK233-2,1° a commercially available expression vector (Pharmacia LKB Biotechnology, Inc., Piscataway, N J). Expression in a lacI q strain is induced by adding IPTG to late log-phase cells to inactivate the lac repressor. The transcription terminator from the rrnB gene 1~prevents read-through transcription and stabilizes the plasmid. The pBR322 origin of replication was replaced with that from a pUC-derived plasmid to augment expression levels. 10 6 S. C. Khani, P. G. Zaphiropoulos, V. S. Fujita, T. D. Porter, D. R. Koop, and M. J. Coon, Proc. Natl. Acad. Sci. U.S.A. 84, 638 (1987). 7 V. S. Fujita, D. J. Thiele, and M. J. Coon, D N A CellBiol. 9, 111 (1990). s D. R. Koop, E. T. Morgan, G. E. Tarr, and M. J. Coon, J. Biol. Chem. 257, 8472 (1982). 9 N. P. Minton, S. P. Chambers, S. E. Prior, S. T. Cole, and T. Gamier, Focus 10, 56 (1988). l0 j. R. Larson, M. J. Coon, and T. D. Porter, J. Biol. Chem. 266, 7321 (1991). u j. Brosius, T. J. Dull, D. D. Sleeter, and H. F. Noller, J. Mol. Biol. 148, 107 (1981).
[11]
EXPRESSION OF MAMMALIAN P450s IN E. coli
111
Culture and Induction
Because plasmids containing the full-length P450 2El cDNA are not stable in recombination-competent strains ofE. coli, all expression experiments utilized strain MV 1304, a r e c A - derivative of JM 105 (United States Biochemical Corp., Cleveland, OH). Overnight cultures in Luria-Bertani medium n containing 40 mg/liter of ampicillin are used to inoculate up to 9 liters of the same medium in 2-liter flasks, which are then shaken vigorously for 4 to 6 hr. It is not necessary to include ampicillin in the preparatory cultures. Cells harboring pCQ3a are grown at 30° until the time of induction, whereas cells containing pKK3a are cultured at 37°. When the cultures reach an A590of 1.0, induction of expression is initiated by raising the temperature to 37° (pCQ3a-containing cells) or adding IPTG to 2 mM final concentration (pKK3a-containing cells). The cells are harvested by centrifugation after 1-2 hr for pCQ3a and after 4-6 hr for pKK3a. Note. Uncontrolled, high level expression of cytochrome P450 is toxic to E. coli, and it can lead to plasmid loss and instability. Thus, it is necessary to closely regulate P450 expression and to carefully maintain cell stocks. With lac promoters (such a s Ptrc on pKK233-2) it is necessary to grow the cells in the presence of high levels of the lacl repressor, either by utilizing lacl q overexpressor strains or by placing the laclq gene on the expression plasmid.
Preparation and Analysis of Subcellular Fractions
To determine the subcellular distribution of the expressed cytochrome P450, inner and outer membrane fractions, cytosol, and periplasm are prepared from induced cells, based on the fractionation procedures of Neu and Heppel, 13 and Ito et al. 14 The periplasmic fraction is prepared by subjecting the cells to osmotic shock.13 Harvested cells are washed 3 times with ice-cold 10 mM Tris-Cl buffer, pH 8.1 (120 ml/liter of culture), to remove media and then resuspended in room temperature 20% sucrose containing 30 mM Tris-C1, pH 8.0 (80 ml/g wet weight). EDTA is added as a 0.5 M solution, pH 8.0, to a final concentration of 1 raM, and the mixture is allowed to stand for 5 to 10 min at room temperature. The cells are pelleted by centrifugation at 13,000 g for 10 min, resuspended in icecold water, 80 mug wet weight, and held on ice for 10 min. The cells are 12T. Maniatis, E. F. Fritsch, and J. Sambrook, "Molecular Cloning: A Laboratory Manual." Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1982. t3 H. C. Neu and L. A. Heppel, J. Biol. Chem. 240, 3685 (1965). 14 K. Ito, T. Sato, and T. Yura, Cell (Cambridge, Mass.) 11, 551 (1977).
112
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pelleted in a final time, and the supernatant, which contains the released periplasm, is collected. Inner and outer membranes and cytosol are isolated by density gradient centrifugation, TMas follows. Cells are pelleted by centrifugation (5000 g for 8 min in a Sorvall GS-3 rotor) and washed by resuspension in 20 mM potassium phosphate buffer, pH 7.4, and repelleting. The cells are taken up in the same buffer containing 5 mM EDTA and 50 mM KCI and lysed by two passes through a French pressure cell at 18,000 psi. Cell debris is removed by centrifugation at 12,000 g for 10 min in a Sorvall SS34 rotor, and the supernatant is layered over a sucrose step gradient, containing 1.5 ml of 70% sucrose, 2.5 ml of 53% sucrose, and 7.0 ml of 15% sucrose, in 15-ml polyallomer tubes. The samples are subjected to centrifugation at 150,000 g in an SW41 rotor for 4 hr at 4°, after which the inner and outer membrane fractions are recovered as bands in the 53% sucrose and at the interface of the 53 and 70% sucrose layers, respectively. The 15% sucrose layer is recovered as the cytosolic fraction. All three fractions are dialyzed at 4° against 20 volumes of 20 mM potassium phosphate buffer, pH 7.4, containing 5 mM EDTA, to remove the sucrose. The protein concentration of each subcellular fraction is determined,15 and the cytochrome P450 content of each fraction is estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by electrophoretic transfer to nitrocellulose and immunodetection with P450 3aspecific antibody. 7 The developed blots are scanned on a densitometer, and the relative P450 content is normalized to the total cellular protein. The bulk of the P450 is found in the inner membrane fraction (approximately 70%), with the remainder in the cytosol. Although 1-2% may be found in the outer membrane fraction, this probably represents contamination with inner membrane. In these cells, cytochrome P450 2El represents approximately 0.3% of the total protein, as determined by immunoquantitation, and approximately 3% of the total (inner and outer) membrane protein, l0
Determination of Enzymatic Activity For the determination of enzymatic activity it is not necessary to separate inner and outer membrane fractions, and thus a simpler procedure to isolate the total membrane fraction is utilized. Induced cells are harvested and lysed with a French press, as described above, and, after 15 p. K. Smith, R. I. Krolm, G. T. Hermanson, A. K. MaUia, F. H. Gartncr, M. D. Provenzano, E. K. Fujimoto, N. M. Goeke, B. J. Olson, and D. C. Klenk, Anal. Biochem. 150, 76 (1985).
[11]
EXPRESSIONOF MAMMALIANP450s IN E. coli
113
removal of the cell debris by low speed centrifugation, the cleared lysate is submitted to ultracentrifugation (142,000 g for 1 hr at 4°, Beckman Type 45 rotor) to sediment the membrane fraction. The membranes are resuspended in the starting volume of 50 mM Tris-acetate buffer, pH 7.4, containing 0.8 M KC1 and 1 mM EDTA, with the use ofa Potter-Elvehjem homogenizer, and repelleted. The washed membranes are resuspended by homogenization in 10 mM Tris-acetate buffer, pH 7.4, containing 1 mM EDTA and 20% glycerol, at a final concentration of approximately 5 mg of protein/ml. To reconstitute activity it is necessary to solubilize the membranes prior to the addition of mammalian NADPH-cytochrome-P450 reductase; simply adding reductase to the resuspended samples is not effective, perhaps because of the lipid composition or the orientation of the membrane vesicles. Membranes are solubilized by the addition of a 12.5% aqueous solution of n-octyl-fl-D-glucopyranoside (Sigma Chemical Co., St. Louis, MO) to a final concentration of 1.25%, with continuous mixing at room temperature. Purified rabbit liver NADPH-cytochrome-P450 reductase 16 is added at a molar ratio of approximately 3 : 1, with respect to P450, and 1,2-dilaurylphosphatidylcholine is added to a final concentration of 30/zg/ ml. The detergent is then removed by dialysis at 4° against 400 volumes of the resuspension buffer overnight. P450 content can be estimated by immunoquantitation or by CO difference spectroscopy. 17 Cytochrome P450 2El enzymatic activity is determined by measuring N-nitrosodiethylamine (NDEA) deethylase activity. 18 Reaction mixtures in 3-ml stoppered glass vials contain 1 mg of protein, 2 mM NDEA, and 2 mM NADPH, in 50 mM potassium phosphate buffer, pH 7.4, in a total volume of 1 ml. After incubation at 37° for 30 min, the reactions are stopped with 0.2 ml of 30% perchloric acid, and the headspace gas is analyzed for ethylene by gas chromatography, as described by Ding and Coon. 18 Enzymatic activity is dependent on added reductase, NADPH, and substrate and is inhibited by P450 3a antibody; membranes from cells containing the vector only, without a P450 2El cDNA insert, do not catalyze NDEA deethylation. Note. The standard detergents used for membrane solubilization in the preparation of P450 from mammalian tissues (Triton X-100, Tergitol, CHAPS, cholate, and deoxycholate) are not effective in the preparation of active P450 2El from bacterial membranes. Although Triton X-100 is commonly used in the solubilization of bacterial membrane proteins (e.g., 16 j. S. French and M. J. Coon, Arch. Biochem. Biophys. 195, 565 (1979). 17 R. W. Estabrook and J. Werringloer, this series, Vol. 52, p. 212. 18 X. Ding and M. J. Coon, Drug Metab. Dispos. 16, 265 (1988).
114
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[11]
B
416
452
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o M
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i
HETEROLOGOUSEXPRESSION
[11]
sis 32 o r chimeric protein constructs) These types of experiments can give insight into the structural basis of P450 substrate specificity, but other expression systems such as yeast, baculovirus, or Escherichia coli may be better suited to obtain the quantity of P450 required for spectral analysis.
Acknowledgments This research was supported by a grant from the National Institutes of Health (GM 37429). B. J. C. is supported in part by a predoctoral fellowship from Merck, Sharp and Dohme Research Laboratories, Rahway, New Jersey. 32 R. L. Lindberg and M. Negishi, Nature (London) 339, 632 (1989).
[11] E x p r e s s i o n o f M a m m a l i a n P 4 5 0 s in Escherichia coli
By TODD D. PORTER and JANE R. LARSON Introduction
Escherichia coliis a desirable organism for the heterologous expression of proteins owing to its ease of manipulation, the availability of a variety of cloning and expression vectors, well-understood genetics, and the low cost of culture. The expression in bacteria of membrane-bound proteins, however, has proved less efficient than that of cytosolic proteins, presumably because of the lack of intracellular membranes into which these hydrophobic proteins can sequester. Whereas an expressed cytosolic protein may constitute up to 30% of the total cellular protein, expressed membrane-bound proteins typically constitute less then 1% of the total cellular protein. Nonetheless, the segregation of these proteins into the inner membrane of the cell allows subcellular fractionation to serve as a convenient and efficient first step in the isolation of the heterologously expressed protein.
Expression Vectors, Culture, and Induction
Vectors We have examined several types of vectors for expression of P450 in E. coli; critical factors for optimal expression are the strength of the promoter and the number of copies of the plasmid in the cell. A strong METHODSIN ENZYMOLOGY,VOL. 206
Copyright© 1991by AcademicPress,Inc. All rightsof reproductionin any formreserved.
[11]
EXPRESSIONOF MAMMALIANP450s IN E. coli
109
promoter, such as the t r p - l a c fusion promoter trc, 1 and a high copy number, as obtained with the origin of replication from the pUC series of plasmids, 2 yield the highest levels of expression. A third factor which appears to be essential to the stable maintenance of the clone is the use of a regulatable promoter, such that expression of the heterologous protein is constitutively suppressed but can be induced when appropriate. We have used both the temperature-sensitive expression vector pCQV2, 3 which utilizes the h rightward promoter and the temperaturesensitive ci857 repressor, and the chemically inducible expression vector pKK233-2, 4 which utilizes the isopropylthiogalactoside-inducible (IPTG) trc promoter. Although we find the trc promoter to be 3-fold stronger than the h promoter, the pCQV2 system offers the advantage of being less expensive to use, as induction of expression is achieved without the use of the relatively expensive chemical, IPTG. This expense becomes relevant when large-scale cultures (200 liters) are anticipated. An additional advantage of the pCQV2 vector is strain flexibility: the ci857 repressor is encoded on the plasmid, and thus the plasmid is not restricted to h lysogens. In this context, it should be noted that the level of expression of a related enzyme, rat NADPH-cytochrome P-450 reductase, has been shown to differ among various strains of E . c o l i : A disadvantage of the pCQV2 vector is the need to grow the cells at 30° to suppress heterologous protein expression while bringing the cells to late log-phase density, and the subsequent need to rapidly shift the temperature to 37° to induce expression. Whereas one-half volume of prewarmed media (at 42°) can be added to standard cultures to quickly raise the temperature, this is not feasible with large cultures. Our experience indicates, however, that P450 expression is not immediately toxic to the cells, and thus a more gradual increase in temperature, as achieved in large-scale fermentors, is not likely to be detrimental. The trc promoter of pKK233-2 offers a higher level of expression than the h promoter, but it suffers the disadvantage of being restricted to strains which contain the l a c l q gene. These strains (such as JM 1052) constitutively overexpress the l a c I repressor, and thus suppress transcription from the trc promoter in the absence of inducer. Induction of expression requires the addition of IPTG to at least 0.5 mM. The two cytochrome P450 expression vectors we have constructed are 1 j. Brosius, M. Ertte, and J. Storella, J. Biol. Chem. 260, 3539 (1985). 2 C. Yanish-Perron, J. Vieira, and J. Messing, Gene 33, 103 (1985). 3 C. Queen, J. Mol. Appl. Gen, 2~ 1 (1983). 4 E. Amann and J. Brosius, Gene 40, 183 (1985). 5 T. D. Porter, T. E. Wilson, and C. B. Kasper, Arch. Biochem. Biophys. 254, 353 (1987).
110
HETEROLOGOUSEXPRESSION
[11]
Eoo RI
Nco I Eco RI
/¢
°''
pCO3a ! Ind III
Pvu I
~('Xho I/Sell I) Hind III FIG. 1. Cytochrome P450 expression plasmids.
shown in Fig. 1. Both plasmids contain a full-length cytochrome P450 2El c D N A 6'7 encoding the rabbit alcohol-inducible form of the cytochrome
(P450 3a). s The presence of an N c o I restriction site at the translation initiation codon (CCATGG) allowed us to conveniently insert the 2El cDNA into an appropriate cloning site in each vector: N c o I for pKK233-2 and B a m H I for pCQV2. In the latter construct, the BarnHI and N c o I ends of the DNA were made flush with mung bean nuclease prior to ligation. Both vectors contain a ribosome binding site upstream of the cloning site. pCQ3a is derived from pCQV2) and thus expression is induced by a temperature shift to inactivate the ci857 repressor. By replacing the pBR322 origin of replication with that from a pUC-derived plasmid, we obtained an 8-fold greater level of expressed protein, as determined by immunoblotting, which we attribute to the 3- to 5-fold higher copy number for plasmids containing this origin. 9 pKK3a is derived from pKK233-2,1° a commercially available expression vector (Pharmacia LKB Biotechnology, Inc., Piscataway, N J). Expression in a lacI q strain is induced by adding IPTG to late log-phase cells to inactivate the lac repressor. The transcription terminator from the rrnB gene 1~prevents read-through transcription and stabilizes the plasmid. The pBR322 origin of replication was replaced with that from a pUC-derived plasmid to augment expression levels. 10 6 S. C. Khani, P. G. Zaphiropoulos, V. S. Fujita, T. D. Porter, D. R. Koop, and M. J. Coon, Proc. Natl. Acad. Sci. U.S.A. 84, 638 (1987). 7 V. S. Fujita, D. J. Thiele, and M. J. Coon, D N A CellBiol. 9, 111 (1990). s D. R. Koop, E. T. Morgan, G. E. Tarr, and M. J. Coon, J. Biol. Chem. 257, 8472 (1982). 9 N. P. Minton, S. P. Chambers, S. E. Prior, S. T. Cole, and T. Gamier, Focus 10, 56 (1988). l0 j. R. Larson, M. J. Coon, and T. D. Porter, J. Biol. Chem. 266, 7321 (1991). u j. Brosius, T. J. Dull, D. D. Sleeter, and H. F. Noller, J. Mol. Biol. 148, 107 (1981).
[11]
EXPRESSION OF MAMMALIAN P450s IN E. coli
111
Culture and Induction
Because plasmids containing the full-length P450 2El cDNA are not stable in recombination-competent strains ofE. coli, all expression experiments utilized strain MV 1304, a r e c A - derivative of JM 105 (United States Biochemical Corp., Cleveland, OH). Overnight cultures in Luria-Bertani medium n containing 40 mg/liter of ampicillin are used to inoculate up to 9 liters of the same medium in 2-liter flasks, which are then shaken vigorously for 4 to 6 hr. It is not necessary to include ampicillin in the preparatory cultures. Cells harboring pCQ3a are grown at 30° until the time of induction, whereas cells containing pKK3a are cultured at 37°. When the cultures reach an A590of 1.0, induction of expression is initiated by raising the temperature to 37° (pCQ3a-containing cells) or adding IPTG to 2 mM final concentration (pKK3a-containing cells). The cells are harvested by centrifugation after 1-2 hr for pCQ3a and after 4-6 hr for pKK3a. Note. Uncontrolled, high level expression of cytochrome P450 is toxic to E. coli, and it can lead to plasmid loss and instability. Thus, it is necessary to closely regulate P450 expression and to carefully maintain cell stocks. With lac promoters (such a s Ptrc on pKK233-2) it is necessary to grow the cells in the presence of high levels of the lacl repressor, either by utilizing lacl q overexpressor strains or by placing the laclq gene on the expression plasmid.
Preparation and Analysis of Subcellular Fractions
To determine the subcellular distribution of the expressed cytochrome P450, inner and outer membrane fractions, cytosol, and periplasm are prepared from induced cells, based on the fractionation procedures of Neu and Heppel, 13 and Ito et al. 14 The periplasmic fraction is prepared by subjecting the cells to osmotic shock.13 Harvested cells are washed 3 times with ice-cold 10 mM Tris-Cl buffer, pH 8.1 (120 ml/liter of culture), to remove media and then resuspended in room temperature 20% sucrose containing 30 mM Tris-C1, pH 8.0 (80 ml/g wet weight). EDTA is added as a 0.5 M solution, pH 8.0, to a final concentration of 1 raM, and the mixture is allowed to stand for 5 to 10 min at room temperature. The cells are pelleted by centrifugation at 13,000 g for 10 min, resuspended in icecold water, 80 mug wet weight, and held on ice for 10 min. The cells are 12T. Maniatis, E. F. Fritsch, and J. Sambrook, "Molecular Cloning: A Laboratory Manual." Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1982. t3 H. C. Neu and L. A. Heppel, J. Biol. Chem. 240, 3685 (1965). 14 K. Ito, T. Sato, and T. Yura, Cell (Cambridge, Mass.) 11, 551 (1977).
112
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pelleted in a final time, and the supernatant, which contains the released periplasm, is collected. Inner and outer membranes and cytosol are isolated by density gradient centrifugation, TMas follows. Cells are pelleted by centrifugation (5000 g for 8 min in a Sorvall GS-3 rotor) and washed by resuspension in 20 mM potassium phosphate buffer, pH 7.4, and repelleting. The cells are taken up in the same buffer containing 5 mM EDTA and 50 mM KCI and lysed by two passes through a French pressure cell at 18,000 psi. Cell debris is removed by centrifugation at 12,000 g for 10 min in a Sorvall SS34 rotor, and the supernatant is layered over a sucrose step gradient, containing 1.5 ml of 70% sucrose, 2.5 ml of 53% sucrose, and 7.0 ml of 15% sucrose, in 15-ml polyallomer tubes. The samples are subjected to centrifugation at 150,000 g in an SW41 rotor for 4 hr at 4°, after which the inner and outer membrane fractions are recovered as bands in the 53% sucrose and at the interface of the 53 and 70% sucrose layers, respectively. The 15% sucrose layer is recovered as the cytosolic fraction. All three fractions are dialyzed at 4° against 20 volumes of 20 mM potassium phosphate buffer, pH 7.4, containing 5 mM EDTA, to remove the sucrose. The protein concentration of each subcellular fraction is determined,15 and the cytochrome P450 content of each fraction is estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by electrophoretic transfer to nitrocellulose and immunodetection with P450 3aspecific antibody. 7 The developed blots are scanned on a densitometer, and the relative P450 content is normalized to the total cellular protein. The bulk of the P450 is found in the inner membrane fraction (approximately 70%), with the remainder in the cytosol. Although 1-2% may be found in the outer membrane fraction, this probably represents contamination with inner membrane. In these cells, cytochrome P450 2El represents approximately 0.3% of the total protein, as determined by immunoquantitation, and approximately 3% of the total (inner and outer) membrane protein, l0
Determination of Enzymatic Activity For the determination of enzymatic activity it is not necessary to separate inner and outer membrane fractions, and thus a simpler procedure to isolate the total membrane fraction is utilized. Induced cells are harvested and lysed with a French press, as described above, and, after 15 p. K. Smith, R. I. Krolm, G. T. Hermanson, A. K. MaUia, F. H. Gartncr, M. D. Provenzano, E. K. Fujimoto, N. M. Goeke, B. J. Olson, and D. C. Klenk, Anal. Biochem. 150, 76 (1985).
[11]
EXPRESSIONOF MAMMALIANP450s IN E. coli
113
removal of the cell debris by low speed centrifugation, the cleared lysate is submitted to ultracentrifugation (142,000 g for 1 hr at 4°, Beckman Type 45 rotor) to sediment the membrane fraction. The membranes are resuspended in the starting volume of 50 mM Tris-acetate buffer, pH 7.4, containing 0.8 M KC1 and 1 mM EDTA, with the use ofa Potter-Elvehjem homogenizer, and repelleted. The washed membranes are resuspended by homogenization in 10 mM Tris-acetate buffer, pH 7.4, containing 1 mM EDTA and 20% glycerol, at a final concentration of approximately 5 mg of protein/ml. To reconstitute activity it is necessary to solubilize the membranes prior to the addition of mammalian NADPH-cytochrome-P450 reductase; simply adding reductase to the resuspended samples is not effective, perhaps because of the lipid composition or the orientation of the membrane vesicles. Membranes are solubilized by the addition of a 12.5% aqueous solution of n-octyl-fl-D-glucopyranoside (Sigma Chemical Co., St. Louis, MO) to a final concentration of 1.25%, with continuous mixing at room temperature. Purified rabbit liver NADPH-cytochrome-P450 reductase 16 is added at a molar ratio of approximately 3 : 1, with respect to P450, and 1,2-dilaurylphosphatidylcholine is added to a final concentration of 30/zg/ ml. The detergent is then removed by dialysis at 4° against 400 volumes of the resuspension buffer overnight. P450 content can be estimated by immunoquantitation or by CO difference spectroscopy. 17 Cytochrome P450 2El enzymatic activity is determined by measuring N-nitrosodiethylamine (NDEA) deethylase activity. 18 Reaction mixtures in 3-ml stoppered glass vials contain 1 mg of protein, 2 mM NDEA, and 2 mM NADPH, in 50 mM potassium phosphate buffer, pH 7.4, in a total volume of 1 ml. After incubation at 37° for 30 min, the reactions are stopped with 0.2 ml of 30% perchloric acid, and the headspace gas is analyzed for ethylene by gas chromatography, as described by Ding and Coon. 18 Enzymatic activity is dependent on added reductase, NADPH, and substrate and is inhibited by P450 3a antibody; membranes from cells containing the vector only, without a P450 2El cDNA insert, do not catalyze NDEA deethylation. Note. The standard detergents used for membrane solubilization in the preparation of P450 from mammalian tissues (Triton X-100, Tergitol, CHAPS, cholate, and deoxycholate) are not effective in the preparation of active P450 2El from bacterial membranes. Although Triton X-100 is commonly used in the solubilization of bacterial membrane proteins (e.g., 16 j. S. French and M. J. Coon, Arch. Biochem. Biophys. 195, 565 (1979). 17 R. W. Estabrook and J. Werringloer, this series, Vol. 52, p. 212. 18 X. Ding and M. J. Coon, Drug Metab. Dispos. 16, 265 (1988).
114
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i
