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[14] E x p r e s s i o n o f M a m m a l i a n C y t o c h r o m e P 4 S 0 E n z y m e s Using Y e a s t - B a s e d Vectors

By F. PETER GUENCERICH, WILLIAM R. BRIAN, MARm-AGNi~SSAm, and JOHN T. ROSS Introduction In recent years the complexity of the cytochrome P450 (P450) gene family has made the definitive assignment of catalytic activities to specific amino acid sequences difficult in many cases. The analysis of structure-function relationships also requires that the catalytic activities of modified proteins be measured. For these reasons it is necessary that artificial expression systems be used to produce proteins corresponding to individual amino acid sequences. When mammalian intrinsic membrane proteins are expressed in bacterial systems proteolysis can be a serious problem 1unless specific constructions are used to direct the proteins. 2,3 Even then, the bacterial systems are devoid of NADPH-cytochrome-P450 reductase, and the expressed P450s would have to be purified and reconstituted. Mammalian cell culture expression systems have been popular, and the technology involving the vectors is reasonably well understood. However, the levels of expression are often very low, precluding catalytic analysis without highly sensitive methods, or the expression is transient. Further, the costs of mammalian cell culture are high and, except for the possibility of bacculovirus-based systems, not suited for large-scale preparation of P450s. The use of yeast as an expression vehicle offers some advantages in the synthesis of large amounts of mammalian P450s without problems associated with proteolysis. Yeast contain their own endoplasmic reticulum, NADPH-cytochrome-P450 reductase, and cytochrome bs, and the endogenous P450s are devoted primarily to sterol oxidation. 4 Although a yeast (Saccharomyces cerevisiae) has been used for the expression of a P450 found in another yeast (Candida tropicalis P450LII),5 this chapter focuses on the expression of mammalian P450s in yeast. I T. D. Porter, T. E. Wilson, and C. B. Kasper, Arch. Biochem. Biophys. 254, 353 (1987). 2 A. L. Shen, T. D. Porter, T. E. Wilson, and C. B. Kasper, J. Biol. Chem. 264, 7584 (1989). 3 j. Ghrayeto, H. Kimura, M. Takahara, H. Hsuing, Y. Masui, and M. Inouye, EMBO J. 3, 2437 (1984). 4 0 . K[ipelli, Microbiol. Rev. 50, 244 (1986). 5 D. Sanglard and J. C. Loper, Gene 76, 121 (1989).

METHODSIN ENZYMOLOGY,VOL. 206

Copyright© 1991by AcademicPress,Inc. All rightsof reproductionin any form reserved.

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Uses o f Yeast-Based S y s t e m s

A comprehensive listing of applications of yeast-based expression systems used to date is presented in Table I. Some notable unique applications will be pointed out. Shimizu et al. 6 were able to prepare enough of each of several mutants of rat P450IA2 to characterize the interactions of ligands using spectral techniques. In most of the cases presented here, precise quantitation of the total amount of P450 was possible using spectral techniques, which is necessary if catalytic rates are to be quantified accurately, especially in making comparisons with mammalian tissues. Studies with human P450IIC proteins led to the view that tolbutamide hydroxylation and S-mephenytoin 4'-hydroxylation are catalyzed by distinct proteins, 7 and Relling et al. 8 have recently provided further evidence to support this view using mammalian cell expression systems. The large-scale production of P450s in yeast is a possibility. In this laboratory and in the work of Ohkawa and Imai, yields of approximately 8 nmol P450/liter have been obtained in shaking cultures, and it would appear that similar levels can be produced in larger fermentors, as judged by our own experience (see below) and that of Renaud et al. 9 Consideration o f Vectors and Yeast Strains

The efficiency of expression of mammalian P450s is a function of the yeast strain, the copy number of the expression plasmid, the promoter, the length of 5'-flanking sequence of the expressed cDNA, the particular amino acid sequence being expressed, and, in the case of fused P450/ NADPH-cytochrome-P450 reductase chimeras, the sequence attached to the P450,1°-16 The various plasmids that have been used are listed in Table I. 6 T. Shimizu, K. Hirano, M. Takahashi, M. Hatano, and Y. Fujii-Kuriyama, Biochemistry 27, 4138 (1988). 7 W. R. Brian, P. K. Srivastava, D. R. Umbenhauer, R. S. Lloyd, and F. P. Guengerich, Biochemistry 28, 4993 (1989). 8 M. V. Relling, T. Aoyama, F. J. Gonzalez, and U. A. Meyer, J. Pharmacol. Exp. Ther. 252, 442 (1990). 9 j._p. Renaud, C. Cullin, D. Pompon, P. Beaune, and D. Mansuy, Eur. J. Biochem. 194, 889 (1990). l0 T. Sakaki, K. Oeda, M. Miyoshi, and H. Ohkawa, J. Biochem. (Tokyo) 96, 167 (1985). H T. Sakaki, K. Oeda, Y. Yabusaki, and H. Ohkawa, J. Biochem. (Tokyo) 99, 741 (1986). 12T. Shimizu, K. Sogawa, Y. Fujii-Kuriyama, M. Takahashi, Y. Ogoma, and M. Hatano, FEBS Lett. 207, 217 (1986). 13 C. Cullin and D. Pompon, Gene 65, 203 (1988). [4 D. Pompon, Fur. J. Biochem. 177, 285 (1988). ]5 V. S. Fujita, D. J. Thiele, and M. J. Coon, DNA Cell. Biol. 9, 111 (1990). 16M. Shibata, T. Sakaki, Y. Yabusaki, H. Murakami, and H. Ohkawa, DNA Cell. Biol. 9, 27 (1990).

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TABLE I MAMMALIANP450 ENZYMESEXPRESSEDIN YEASTSYSTEMS P450 enzyme Rat P450IA1 Rat P450IAI Rat P450IAI, P450IA2, and chimeric hybrids Rat P450IA1, NADPHcytochrome-P450 reductase, and fused chimera Rat P450IA2 and derivatives

Vector pAAH5 (alcohol dehydrogenase) pAAH5 pAAH5 pAAH5

pAM82 (acid phosphatase)

Rat P450IVA1 Rat P450IIB1

pAAH5 pmA56 (yeast A D C I )

Rat P450IIC11

pYcDE2 ( A D H prorooter, C Y C I terminator) pAAH5 pYe DP1, 10 (phosphoglycerate kinase, GALIO gene, iso-l-cytochrome c) PYe DPI,10

Mouse P450IA1 Mouse P450IAI, P450IA2, and hybrids

Rabbit P450IA1, P450IA2, and hybrid (also chimeras with mouse P450IAI) Rabbit P450 pHP3

pAAH5

Rabbit P450 (laurate co-l) pAH3P2 Rabbit P450 (testosterone 16a-hydroxylase) pAHF3 Rabbit P450 pH P2-1 and P450 pHP3 chimeras

pAAH5

Rabbit P450IIE1

YEpl3 (CUP1, metallothionein) pAAH5

Rabbit P450 kal, P450 ka2

Substrate

Ref.

Benzo[a]pyrene, 7-ethoxycoumafin 7-Ethoxycoumarin, acetanilide 7-Ethoxycoumarin, acetanilide, benzo[a]pyrene 7-Ethoxycoumarin

17/3-Estradiol, acetanilide, benzphetamine, 7-ethoxycoumarin, zoxazolamine Lauric acid Benzyloxyresorufin, cyclophosphamide Testosterone, benzphetamine, 7-ethoxycoumarin Benzo[a]pyrene Ethoxyresorufin

Benzo[a]pyrene, acetanilide, 7-ethoxycoumarin, ethoxyresorufin, progesterone, acetanilide Benzphetamine, aminopyrine, l-nitropropane Capric acid, lauric acid

a, b c d e, f

g, h, i, j, k, 1

m n o

p q

r, s

t, u v, w

pAAH5

Testosterone, progesterone

v, w

pAAH5

Caprylic acid, capric acid, lauric acid, benzphetamine, aminopyfine, 1-nitropropane N-Nitrosodiethylamine, aniline Prostaglandin (lauric acid) A1

u, x

y z

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TABLE I (continued) P450 enzyme

Vector

Substrate

Bovine P450XVIIA and fused NADPH-cytochromeP450 reductase chimeras Human P450IIC9 Human P450IIC9 Human P450IIC10

pAAH5

Progesterone, 17ahydroxyprogesterone

pAA 7 (Gal7) pAAH5 pAAH5

Human P450IIC9

pAAH5

Human P4501IIA4

PYe DPI,10

Human P450IIIA4

pAAH5

Mephenytoin Mephenytoin Tolbutamide, hexobarbital, tienilic acid Tolbutamide, hexobarbital, tienilic acid Nifedipine, quinidine, troleandomycin, erythromycin See Table II

Ref. aa, bb

cc dd ee

ff gg

hh

a T. Sakaki, K. Oeda, M. Miyoshi, and H. Ohkawa, J. Biochem. (Tokyo) 98, 167 (1985). b K. Oeda, T. Sakaki, and H. Ohkawa, DNA 4, 203 (1985). c T. Sakaki, K. Oeda, Y. Yabusaki, and H. Ohkawa, J. Biochem. (Tokyo) 99, 741 (1986). d T. Sakaki, M. Shibata, Y. Yabusaki, and H. Ohkawa, DNA 6, 31 (1987). e H. Murakami, Y. Yabusaki, and H. Ohkawa, DNA 5, 1 (1986). f H. Murakami, Y. Yabusaki, T. Sakaki, M. Shibata, and H. Ohkawa, DNA 6, 189 (1987). g T. Shimizu, K. Sogawa, Y. Fujii-Kuriyama, M, Takahashi, Y. Ogoma, and M. Hatano, FEBS Lett. 207, 217 (1986). h T. Shimizu, K. Hirano, M. Takahashi, M. Hatano, and Y. Fujii-Kuriyama, Biochemistry 27, 4138 (1988). i T. Shimizu, A. J. Sadeque, M. Hatano, and Y. Fujii-Kuriyama, Biochim. Biophys. Acta 995, 116 (1989). J H. Furuya, T. Shimizu, M. Hatano, and Y. Fujii-Kuriyama, Biochem. Biophys. Res. Commun. 160, 669 (1989). k H. Furuya, T. Shimizu, K. Hirano, M. Hatano, Y. Fujii-Kuriyama, R. Raag, and T. Poulos, Biochemistry 28, 6848 (1989). t A. J. Sadeque, T. Shimizu, K. Hirano, M. Hatano, and Y. Fujii-Kuriyama, Inorg. Chim. Acta 153, 161 (1988). m j. p. Hardwick, B.-J. Song, E. Huberman, and F. J. Gonzalez, J. Biol. Chem. 262, 801 (1987). S. M. Black, S. Ellard, R. R. Meehan, J. M. Parry, M. Adesnik, J. D. Beggs, and C. R. Wolf, Carcinogenesis 10, 2139 (1989). o S. Hayashi, K. Morohashi, H. Yoshioka, K. Okuda, and T. Omura, J. Biochem. (Tokyo) 103, 858 (1988). P S. Kimura, H. H. Smith, O, Hankinson, and D. W. Nebert, EMBO J. 6, 1929 (1987). q C. Cullin and D. Pompon, Gene 65, 203 (1988). • D. Pompon, Eur. J. Biochem. 177, 285 (1988). s D. Pompon and A. Nicolas, Gene 83, 15 (1989). t y. Imai, J. Biochem. (Tokyo) 101, 1129 (1987). u y. Imai, J. Biochem. (Tokyo) 103, 143 (1988). v y. Imai and M. Nakamura, FEBS Lett. 234, 313 (1988). Y. Imai and M. Nakamura, Biochem. Biophys. Res. Commun. 158, 717 (1989). x T. Uno and Y. Imai, J. Biochem. (Tokyo) 106, 569 (1989).

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The first system to be used was the pAAH5 vector, based on an alcohol dehydrogenase promoter and terminator.17 This has been used in the expression of a number of different P450s, including several in this laboratory. Other plasmids containing constitutive and inducible promoters have been used for the expression of some of the P450s. Shimizu e t al. 12 found an acid phosphatase promoter system to be considerably more efficient (than the alcohol dehydrogenase-based pAAH5) for the expression of rat P450IA2. Pompon 14 utilized a phosphoglycerate kinase-based promoter and reported a substantial decrease in the rabbit P450IA2 expression level with increasing length of the cDNA 5' noncoding sequence retained in the expression vector. Sakaki e t al. ~8 replaced the 5' upstream sequence of bovine P450XVIIA with the sequence AAGCTTAAAAAAATG, which contains a consensus sequence important for highly efficient translation in S. c e r e v i s i a e ; 19 the substitution resulted in a 2-fold increase in the level of the mRNA but no change in the level of the protein or its activity. Fujita e t al. ~5 reported that the amount of rabbit P450IIE1 protein expressed could be substantially increased when a metallothionein-based (CUP1) YEpI3 promoter was used and induced by short-term incubation with copper (although comparison of the amounts of holoenzyme or active enzyme between the two systems was not reported). Different yeast strains have also been considered. In our experience 17 G. Ammerer, this series, Vol. 101, p. 192. 18 T. Sakaki, M. Shibata, Y. Yabusaki, H. Murakami, and H. Ohkawa, DNA 8, 409 (1989). 19 R. Hamilton, C. K. Watanabe, and H. A. De Boer, Nucleic Acids Res. 15, 3581 (1987).

YV. S. Fujita, D. J. Thiele, and M. J. Coon, DNA CellBiol. 9, 111 (1990). z N. Yokotani, R. Bernhardt, K. Sogawa, K. Kusunose, O. Gotoh, M. Kusunose, and Y. Fujii-Kuriyama, J. Biol. Chem. 264, 21665 (1989). a a T. Sakaki, M. Shibata, Y. Yabusaki, H. Murakami, and H. Ohkawa, DNA 8, 409 (1989). bb M. Shibata, T. Sakaki, Y. Yabusaki, H, Murakami, and H. Ohkawa, DNA Cell Biol. 9, 27 (1990). cc T. Yasumori, N. Murayama, Y. Yamazoe, Y. Nogi, T. Fukasawa, and R. Kato, Mol. Pharmacol. 35, 443 (1989). dd S. Ohgiya, M. Komori, T. Fujitani, T. Miura, N. Shinriki, and T. Kamataki, Biochem. Int. 13, 429 (1989). ee W. R. Brian, P. K. Srivastava, D. R. Umbenhauer, R. S. Lloyd, and F. P. Guengerich, Biochemistry 28, 4993 (1989). W. R. Brian, M.-A. Sad, M. Iwasaki, T. Shimada, L. S. Kaminsky, and F. P. Guengerich, Biochemistry 29, 11280 (1990). g8 j..p. Renaud, C. Cullin, D. Pompon, P. Beaune, and D. Mansuy, Eur. J. Biochem. 194, 889 (1990). hh p. K. Srivastava, C-H. Yun, P. H. Beaune, C. Ged, and F. P. Guengerich, Mol. Pharmacol., in press.

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similar results have been obtained using S. cerevisiae D12 (from the Genex Corp., Gaithersburg, MD) and AH22 cells (from Drs. Y. Yabusaki and H. Ohkawa, Sumitomo Chemical Co., Takarazuka Hyogo, Japan). It should be pointed out that the literature suggests that different yeasts vary in their levels of NADPH-cytochrome-P450 reductase; thus, some systems require the addition of reductase to yeast microsomes,9'1°,~5,2° whereas others give optimal activity without the addition of exogenous re-

ductase.7,21 In summary, several different plasmids and yeasts have been utilized for expression. Our own experience would suggest that the use of plasmid pAAH5 and AH22 cells is a reasonable starting point, based on the amount of previous work with this system by several laboratories. However, other systems might be expected to have similar probability for success with a new cDNA to be expressed, and it is not possible to recommend a single system that will be optimal for all purposes. In the future, more efficient systems will probably become available.

Catalytic Assays Different approaches have been used for incubations. In some studies whole yeast cells have been used) 1,16,18,22 This approach is technically simple although long incubation times are required and caveats about uptake of substrates into cells and side reactions must be considered. Our own experience with the sensitivity in such systems has not been particularly satisfactory. Another approach involves the preparation of microsomes, which can be used in lieu of liver preparations. In our opinion, this is the preferred approach, since in many cases (or at least with the pAAH5 vector and S. cerevisiae D12 or AH22 cells) the levels of NADPH-cytochrome-P450 reductase and cytochrome b5 and the membrane environment reflect the hepatic microsomal milieu. Surrogate oxygen donors such as cumene hydroperoxide can be used in lieu of NADPH,~4.21 if there are reservations about the functional coupling with yeast NADPH-cytochrome-P450 reductase. The third option is to partially or completely purify the P450 from the yeast microsomes and reconstitute a catalytic system with added NADPH-cytochrome-P450 reductase, phospholipid, and possibl3, cytochrome bs. This approach has been used in the

20 T, Yasumori, N. Murayama, Y. Yamazoe, Y. Nogi, T. Fukasawa, and R. Kato, Mol. Pharmacol. 35, 443 (1989). 21 W. R. Brian, M.-A. Sail, M. Iwasaki, T. Shimada, L. S. Kaminsky, and F. P. Guengerich, Biochemistry 29, 11280 (1990). 22 T. Sakaki, M. Shibata, Y. Yabusaki, and H. Ohkawa, DNA 6, 31 (1987).

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TABLE II SUBSTRATES FOR YEAST P450IIIA4 Drugs Nifedipine and 18 other dihydropyridines, including nitrendipine, nimodipine, niludipine, nisoldipine, nicardipine, felodipine, and ( + )- and ( - )-Bayer K8644 (pyridine formation and other reactions) ~'b (R)- and (S)-Warfadn (10-hydroxylation and 9,10-dehydrogenation of the respective enantiomers) ~ Erythromycin (N-demethylation) a Quinidine (N-oxygenation and 3-hydroxylation) ~ Lovastatin (6'fl-, Y'-, and 6'-exo-methylene hydroxylations) c Steroids Testosterone (6fl-hydroxylation)a Cortisol (6fl-hydroxylation) a 17fl-Estradiol (2-hydroxylation) ~ 17a-Ethynylestradiol (2-hydroxylation) a Gestodene (suicide inactivation-acetylenic group) a,d Dehydroepiandrosterone 3-sulfate (16a-hydroxylation) ~ Carcinogens Aflatoxin B1 (8,9-epoxidation) a Aflatoxin Gl (9,10-epoxidation) a Sterigmatocystin (1,2-epoxidation) ~ ( + )- and ( - )-7,8-Dihydroxy-7,8-dihydrobcnzo[a]pyrene (9,10-epoxidation) a 3,4-Dihydroxy-3,4-dihydrobenz[a]anthracene ~ 3,4-Dihydroxy-3,4-dihydro-7,12-dimethylbenz[a]anthracene ~ 9,10-Dihydroxy-9,10-dihydrobenzo[b]fluoranthenea 6-Aminochrysene ~ Tris(2,3-dibromopropyl) phosphate a a W. R. Brian, M.-A. Sari, M. Iwasaki, T. Shimada, L. S. Kaminsky, and F. P. Guengerich, Biochemistry 29, 11280 (1990). b F. P. Guengerich, W. R. Brian, M. Iwasaki, M.-A. Sad, and C. B~i~trnhielm, J. Med. Chem. 34, 1838 (1991). c R. W. Wang, P. H. Kari, A. Y. H. Lu, P. E. Thomas, F. P. Guengerich, and K. P. Vyas, submitted. e F. P. Guengerich, and T. Shimada, Chem. Res. Toxicol. (in press).

work of I m a i 23-26 and in a situation in this laboratory with human P450IIIA4 where it was found to be more reliable than measurement of activity in microsomes) 1 Although the purification of P450s from yeast is easier than liver, however, this method does involve more effort and also involves the use of an environment that is probably more artificial than seen in the microsomes. 23 y . 24 y . 25 y . 26 y .

Imai, J. Biochem. (Tokyo) 101, 1129 (1987). Imai, J. Biochem. (Tokyo) 103, 143 (1988). Imai and M. Nakamura, FEBS Lett. 234, 313 (1988). Imai and M. Nakamura, Biochem. Biophys. Res. Commun. 158, 717 (1989).

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In general, rates of catalysis are similar to or higher than those seen in liver microsomes, and similar assays can be employed [i.e., high-performance liquid chromatography (HPLC) with detection by UV, fluorescence, or radiometry, extraction assays, spectrophotometric assays] (Table II). E x p e r i m e n t a l Details

Yeast Strains In our work on expression of the human MP-8 cDNA clone in Saccharomyces cerevisiae, the D12 strain [leu- (cir+); gift from Genex Corp., Gaithersburg, MD] was used successfully.7After that work was completed we obtained the AH22 strain [a, leu2-3, leu2-112, his 4-519, can 1, (cir ÷) (rho°); gift from Drs. Y. Yabusaki and H. Ohkawa (Sumitomo Chemical Co., Takarazuka Hyogo, Japan)], which has been used by many other researchers for expression of P450 enzymes in y e a s t . 6,7,l°'ltA6'la'2t'22,24-3° Expression of the MP-8 cDNA clone in the AH22 strain showed a 1.5-fold increase in the amount of P450 produced. Yeast strains D12 and AH22 were maintained on YPD medium [2% Bactopeptone (w/v, Difco, Detroit, MI), 1% yeast extract (w/v, Difco), and 2% glucose (w/v)]. Transformed yeast were maintained on minimal SD medium [0.67% yeast nitrogen base without amino acids (w/v, Difco), 2% glucose (w/v), and 20/zg histidine/ml (w/v)].

Expression Vector Construction The pAAH5 yeast expression vector (gift from Dr. B. Hail, University of Washington, Seattle, WA) used in work in this laboratory utilizes the alcohol dehydrogenase 1 promoter and terminator and contains the leu2 gene, allowing selection for transformants by leucine complementation.7 Details of protocols used to insert the human P450 cDNA clones in the pAAH5 vector are found elsewhere.7 We have used only the ATG start site for the protein at the 5' end of the inserted sequence.

27 H. Murakami, Y. Yabusaki, and H. Ohkawa, DNA 5, 1 (1986). H. Murakami, Y. Yabusaki, T. Sakaki, M. Shibata, and H. Ohkawa, D N A 6, 189 (1987). 29T. Shimizu, A. J. Sadeque, M. Hatano, and Y. Fujii-Kuriyama,Biochim. Biophys. Acta 995, 116 (1989). 30 S. Ohgiya, M. Komori, T. Fujitani, T. Miura, N. Shinriki, and T. Kamataki, Biochem. Int. 13, 429 (1989).

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Yeast Transformation Yeast are transformed using the general spheroplast method of Beggs. 31 A single colony of yeast strain D12 or AH22 is grown 16 hr to saturation in 5 ml YPD medium (50-ml flask) at 30°, and 50 ml of YPD (250-ml flask) is then inoculated with 1.5 ml of the saturated culture and shaken at 30° for approximately 2 hr. The growth of the culture is measured by light scattering at 600 nm (A~00, Varian 635 M spectrophotometer, Varian, Walnut Creek, CA). When the A600 value reaches a value of 0.4 to 0.6, cells are pelleted by centrifugation at 450 g for 10 rain and the supernatant decanted. Cells are washed with 25 ml of 1.2 M sorbitol, pelleted as before, and resuspended in 9 ml of 1.2 M sorbitol. The yeast cell walls are enzymatically removed using lyticase (Sigma Chemical Co., St. Louis, MO; Catalog No. L8137). Similar enzyme preparations are available from other suppliers, but lyticase is chosen because it can be obtained in relatively high purity and at reasonable cost for use in yeast transformation. Lyticase, dissolved in water to 2 mg/ml and stored at - 20° until use, is added to the cell suspension to a final concentration of 45/~g/ml. Cells are incubated at room temperature for 12-15 min for spheroplast formation, monitored according to the following protocol: to each of five clear glass test tubes is added 1 ml of water containing 0.2% (w/v) sodium dodecyl sulfate (SDS). As a control, a 50-/~1 aliquot of cells is added to one tube (without lyticase), resulting in a cloudy suspension. Following the addition of lyticase the cells are mixed frequently by inversion, and at 6, 8, 10, and 12 min 50-/~1 aliquots of the cell suspension are removed and added to individual tubes containing the SDS solution. The spheroplasts lyse in this solution so that as spheroplast formation proceeds the cloudiness lessens. After 12-15 min, the cloudiness visibly decreases to less than 50% of the control. At this point the cells (spheroplasts) are very fragile and should be handled delicately during the following transformation steps. Vortex mixing should be avoided. Spheroplasts pellet loosely following a low speed centrifugation at 250 g for 3 rain and tall up the tube wall. The supernatant is decanted carefully, and the pellet is resuspended in 1 ml of 1.2 M sorbitol by gently shaking the tube. An additional 9 ml of 1.2 M sorbitol is then added. Resuspension in this fashion, rather than adding 10 ml at once, results in less cell clumping. Spheroplasts are pelleted as before and washed with 10 ml of 10 mM Tris-HCl buffer (pH 7.4) containing 1.2 M sorbitol and 10 mM CaC12, added in sequential 1-ml and 9-ml aliquots. 31 j. D. Beggs, Nature (London) 295, 104 (1978).

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After centrifugation, spheroplasts are resuspended in 500/zl of the same solution, and 100-/~1 aliquots are transferred to sterile tubes. Approximately 1/~g of pAAH5 plasmid containing the P450 cDNA is added to 100 /~1 of spheroplasts. For controls, 1/zg of pAAH5 plasmid alone (no P450 cDNA) or an equivalent volume of water (no DNA) is added to spheroplasts. After DNA addition the spheroplasts are incubated at room temperature for 15 min. To each 100-/zl aliquot is added 900/.d of 10 mM TrisHCI buffer (pH 7.4) containing 20% polyethylene glycol 8000 (Sigma) and 100 mM CaC12, and incubation is continued an additional 15 min. Aliquots are then centrifuged at 250 g for 3 min, and the supernatant is very carefully withdrawn (the pellet is very loose). Spheroplasts are resuspended in 100 /xl of 1.2 M sorbitol, containing I0 mM CaC12 and 20/~g leucine/ml, plus 50/~1 of YPD containing 1.2 M sorbitol. After a 20-min incubation at room temperature, 800/zl of 1.2 M sorbitol is added. For plating, 200/,d of the transformed spheroplasts is mixed with 15 ml of regeneration medium [0.67% yeast nitrogen base without amino acids (w/v), 2% glucose (w/v), and 1.2 M sorbitol containing 3% agar (kept at 52°)] in 100-mm petri dishes. Plates are inverted and incubated at 30° for 4-5 days. After 1 day the plates are wrapped with Parafilm to reduce moisture loss. Growth of transformants is initially detected by the appearance of cream-colored circular colonies, most of which are embedded in the agar. As the colonies grow larger, those embedded in the agar become ovalshaped. Typical transformations result in approximately 100 transformants//.Lg of P450-containing DNA. The AH22 strain gave slightly more transformants than the DI2 strain.

Colony Screeningfor P450 Expression Individual transformed colonies are removed from the regeneration plates by coring the agar with the small end of a sterile disposable pipette. The agar plugs are added to 1 ml of SD medium in sterile test tubes and shaken for 16 hr at 30°. In this time most of the colonies grow to saturation. Colonies are grown in the minimal SD medium, rather than complex YPD medium, to maintain selective pressure for maximum retention of the plasmid. Prior to screening, each culture arising from the single colonies is streaked onto SD plates for storage (referred to as master plates). Colonies are first screened for high levels of P450 protein expression by immunoblot analysis. The 1-ml cultures are centrifuged at 6500 g for 10 min (23°), and the pellets are washed with 5 ml of water and resuspended in 1 ml of water. Only cultures that grow to saturation are analyzed, and it is assumed that cell mass is equivalent for all cultures. Sodium dodecyl

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HETEROLOGOUS EXPRESSION

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sulfate-polyacrylamide gel electrophoresis and immunoblot analysis are done as described elsewhere. 7,32,33 Briefly, the cell suspension is mixed with 1/2 volume of solubilization buffer containing 0.2 M Tris-HCl (pH 6.8), 30% (v/v) glycerol, 3% (w/v) SDS, 0.0025% (w/v) pyronin Y, and 2.1 M 2-mercapto-ethanol and heated at 100° for 3 min. This protocol effectively extracts the P450 protein from the yeast cells. Samples are centrifuged at 2000 g for 5 min to pellet debris, and the supernatant is loaded into a 7.5% (w/v) acrylamide gel containing a stacking gel. Following electrophoresis, proteins are transferred to nitrocellulose and blotted with rabbit polyclonal antibodies against the particular human P450 being expressed. 7,32Colonies exhibiting the highest amounts of P450 protein are then screened by spectral analysis. Yeast cells from the master plates corresponding to the selected colonies are grown at 30° to saturation in 50 ml of SD medium (250-ml flasks). Cells from the cultures are collected by centrifugation for I0 min at 8000 g and are washed with 50 ml of water and resuspended in 0.1 M potassium phosphate buffer (pH 7.4), typically giving a concentration of 1.5 x 109 to 2 x 109 cells/ml, determined by manual counting with a hemocytometer. Reduced Fe2+-CO versus Fe 2+ P450 spectra of intact yeast cells are obtained for each culture as described 2°,34,35using a Cary 219 spectrophotometer (Varian, Walnut Creek, CA) in the automatic baseline correction mode. The addition of detergents and glycerol in the solubilization buffer is useful in reducing light scattering in these assays [e.g., 20% glycerol, 0.5% sodium cholate, and 0.4% Emulgen 913 (Kao Atlas,Tokyo)]. Care must be taken to obtain accurate spectra in the concentrated suspension of cells. From the colonies exhibiting high levels of P450 expression in this round of screening, one is selected as the source of expressed P450 for enzymatic analysis. Yeast Growth

No significant difference is found in the amount of P450IIC10 (P450TB) expressed in AH22 yeast when YPD or SD medium is used for growth (expressed on a per liter culture basis). However, in the expression of P450IIIA4 (P450NF), approximately 5 times more P450IIIA4 accumulates in transformed yeast grown in SD medium as compared to growth in YPD medium (expressed on a per liter culture basis). Further studies showed that the level of expression is similar regardless of whether yeast is grown 32 U. K. Laemmli, Nature (London) 227, 680 (1970). 33 F. P. Guengerich, P. Wang, and N. K. Davidson, Biochemistry 21, 1698 (1982). T. Omura and R. Sato, J. Biol. Chem. ?,39, 2370 (1964). 3s K. Oeda, T. Sakaki, and H. Ohkawa, DNA 4, 203 (1985).

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in SD medium or an SD medium supplemented with amino acids (except Leu) with the pH adjusted to 7 with potassium phosphate buffer. Two-liter Erlenmeyer flasks containing 1 liter of SD medium are inoculated with yeast and shaken at 150 rpm at 30° in New Brunswick shakers (New Brunswick Scientific, Edison, N J). Yeast inoculum for the l-liter cultures is prepared in two steps. A single colony from the master plate is transferred to 4 ml of SD medium. This is grown for 16 hr at 30° to saturation, and transferred to 80 ml of the same medium. The culture is grown for 10 hr, and then 10 ml is used to inoculate 1 liter of the same medium. The l-liter cultures typically require 14 hr to reach an A600 value of 1.6 to 1.8, at which time the cells are harvested. Growth is monitored closely at the l-liter stage, as it was found that exceeding an A~0 value of 1.8 greatly decreases the efficiency of cell lysis for microsome preparation. Yeast are harvested by centrifugation at 5000 g for 20 min in l-liter bottles. Cells are pooled, washed with approximately 1/20 volume of water, and then used immediately for microsome preparation or stored at - 2 0 °. Microsome Preparation

Yeast microsomes are isolated by an adaption of the method of Oeda et al. 35 The major difficulty in the process is in breaking the cells. Initially several methods were tried, alone or in combination, including mechanical disruption using glass beads, sonication, homogenization with a Teflon pestle, French press, and commercial homogenization mill. Only a small percentage of cells are broken using these methods. However, a relatively inexpensive enzyme preparation, termed yeast lytic enzyme, may be obtained from ICN Biochemicals (Cleveland, OH; medium purity, Catalog No. 152270) and can be used for such large-scale work. This enzyme, isolated from Arthrobacter luteus, contains /3-1,6-glucanase activity, which degrades yeast cell walls without disrupting membranes. For enzyme treatment, the yeast cell pellet is suspended in 10 mM Tris-HCl buffer (pH 7.5) containing 2 M sorbitol, 0.1 mM dithiothreitol, 0.1 mM EDTA, 0.4 mM phenylmethylsulfonyl fluoride, and 5 mg yeast lytic enzyme/ml (I0 ml buffer/liter of original culture). Cells are then shaken gently for 1 hr at 30°. After I hr, cells (spheroplasts) are collected by centrifugation at 120 g for 10 min, washed with ice-cold I0 mM Tris-HCl buffer (pH 7.5) containing 0.65 M sorbitol, 0.1 mM dithiothreitol, 0.1 mM EDTA, and 0.4 mM phenylmethylsulfonyl fluoride, and resuspended in the same buffer (30 ml buffer/liter of original culture). Spheroplasts are lysed by sonication using four to six 15-sec bursts with a 0.5-inch probe of a Sonifier Cell Disruptor (Model W185; Heat Systems-Ultrasonics, Plainview, NY) at 140 W. The

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spheroplast suspension is kept on ice, and adequate intervals are maintained between bursts to minimize temperature increases. Lysis is monitored by light microscopy. Treating cells with yeast lytic enzyme, followed by sonication, results in breakage of greater than 95% of the cells. As stated previously, to obtain this efficiency it is important to harvest yeast before growth exceeds an A600 value of 1.8. The sonicated suspension is centrifuged at 10,000 g for 30 min, and then the supernatant is centrifuged at 125,000 g for 90 min to pellet microsomes. In our original protocol 7 the microsomal pellet is washed and then resuspended in 10 mM Tris-acetate buffer (pH 7.4) containing 1 mM EDTA and 20% glycerol (v/v). Homogenizing the microsomal pellet in the sonication buffer without washing gives similar results and is faster. The amount of P450 contained in the microsomes is estimated by the reduced CO difference spectra, based on the method of Omura and Sato 34 (see above). Endogenous yeast P450s account for less than 10% of the total P450 in yeast transformed with P450 cDNAs in our experience. Microsomes are used immediately or aliquoted and stored at - 8 0 ° until use. The human P450IIC10 and P450IIIA4 activities appear to be more susceptible to inactivation by freeze-thawing in yeast microsomes than human liver microsomes.

Fermentation Scale-up Strain Expansion and Media Preparation. The initial scaleup into production fermentor systems utilized the Saccharomyces cereoisiae strain DI2 transformed with the cytochrome P450 cDNA clone pAAH5/NF 25 (P450IIIA4) or pAAH5/MP8 (P450IIC 10) and expanded from frozen slants under 15% glycerol. Seed expansion for aseptic introduction into the production fermentor employed revival of the frozen slant by warming to room temperature and isolation streaking onto an SD (His-) agar plate. After a 72-hr incubation period at 30°C, single mature colonies are selected to inoculate each of two first-stage seed flasks containing 50 ml of presterilized SD broth (His-) (in 250-ml baffled shake flasks). The first-stage seed is incubated at 30° and 200 rpm on a floor shaker (Model G-25, New Brunswick Scientific Co., Edison, NJ) for 18 hr prior to aseptic transfer into two second-stage seed flasks, each containing 2 liters of SD broth (His +) in 4-liter baffled Erlenmeyer flasks. After 24 hr of growth at 30° and 200 rpm, the second-stage seed flasks are aseptically pooled into a presterilized 6-liter glass transfer vessel and immediately inoculated into the production fermentor via a MasterFlex peristaltic pump (Cole-Parmer, Chicago, IL). Production Fermentor Preparation. The production fermenter used is

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a 110-liter fermentor manufactured by New Brunswick Scientific (Edison, N J) capable of continuous or semicontinuous fermentation modes. General fermentation practices for preparation, batching, and media sterilization are briefly described as follows: The fermentor is batched with 85 liters of tap water, operationally tested and presterilized at 121° for 2 hr, cooled, and held at 17 psi head pressure. Connections and steam-sealed articulations are checked for leaks using a bubble detection method. Inlet and exhaust filters (0.22/xm, hydrophobic) are bench integrity tested using a forward-flow/pressure hold method prior to installation. The sterile water is dumped from the fermentor, and the vessel is then rinsed and filled to operating volume with reverse osmosis deionized water. Nonreactive, nonlabile media components are added to the agitated vessel, and the fermentor and contents are heated to 121° for 45 min, cooled to operating temperature, and specific parameters (agitation, sparge, etc.) are set. Heat-sensitive materials such as glucose and amino acids are separately sterilized and added to the fermentor after cool-down via a peristaltic pump through a steam-sealed inoculation port. A positive back-pressure is maintained on the vessel at all times to discourage contamination. The sterile medium is sampled via a steam-sealed sample port after sterilization, and inoculated onto a TSAG (trypticase soy agar plus glucose) plate to verify sterility. The uninoculated vessel is additionally sampled at 24 hr postmedium sterilization and microscopically examined for sterility. The production medium contains 6.7 g yeast nitrogen base/liter without amino acids (Difco, Detroit, MI), 20 g anhydrous glucose/liter (J. T. Baker, Phillipsburg, N J), 20/zg histidine base/ml (Aldrich Chemical, Milwaukee, WI), and 68 liters reverse osmosis deionized water. Sterile (autoclaved) Dow Corning type A silicone-based antifoam (Dow Corning, Midland, MI) is also added to the production medium (to 0.1%, v/v) to inhibit foam formation. To prevent sugar caramelization and possible degradation of the amino acid, glucose and histidine are dissolved in water, filter sterilized, and aseptically added to the steam-sterilized basal medium. The pH of the production medium is adjusted in situ to 6.98 using 0.2 M K2HPO 4 after the addition of glucose and histidine. No pH control is employed during the course of the fermentation. Growth in Production Fermentor. A 110-liter stainless steel fermentor containing 68 liter of medium is batched and sterilized as previously described. The fermentor is inoculated with 3.4 liters (5.0%, v/v) of exponentially growing shake flask seed culture (Ar00 0.50) and fermented for 22.5 hr to an Ar00 value of 1.6. The temperature is controlled at 30°. The culture is aerated with 2.50 standard cubic feet per minute (SCFM) air with a back-pressure of 5.0 psi, and 250 rpm agitation providing a tip speed of

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340 ft/min and an oxygen transfer rate (OTR) of approximately 60 mM O 2/ liter/hr, thus ensuring thatthe culture remains aerobic (i.e., culture fluid dissolved oxygen -> 70%). On-line monitoring and microcomputer data logging (IBM-PCAT) are employed for observation and evaluation of culture dynamics. Dissolved oxygen is monitored using a polarographic 02 sensor (Instrumentation Lab Inc., Lexington, MA), and pH is monitored using an Ingold pressurized combination probe (Ingold, Andover, MA). Fermentor head space CO2 and 02 are monitored "on line" using a Model 703D infrared analyzer (Infrared Industries, Santa Barbara, CA) and Beckman Model 755 Paramagnetic analyzer (Model 755, Fullerton, CA), respectively. A600 and packed cell volumes (percent packed cells at 2600 g for l0 min, 23°) are assessed at 2-hr intervals via aseptic sampling and off-line monitoring. Biomass Separation and Cell Disruption. At optimum harvest time, whole broth is pressure fed from the fermentor (10 psi) to a Sepa vertical bowl semicontinuous refrigerated centrifuge (12,000 g), and the cell pellet recovered (pellet weight 375 g). Cell wall disruption is facilitated by resuspension of the cell paste (1 : 2, w/v) in lysis buffer [10 mM Tris-HCl buffer (pH 7.5), containing 2 M sorbitol, 0.1 mM dithiothreitol, 0.1 mM EDTA, 0.4 mM phenylmethylsulfonyl fluoride, and 4.6 mg (w/v) ICN yeast lytic enzyme/ml] incubation on a rotary shaker (30°, 1 hr, 200 rpm), and recentrifugation at low speed (3000 g). The resultant supernatant is discarded, and the cells are resuspended in 2:1 (v/w) buffer (less lytic enzyme), and broken using a Bead Beater with an ice/sodium chloride cooling jacket (Biospec Products, Bartlesville, OK) and a 1 : 1 distribution of 0.5-mm glass beads and cell suspension. Disruption requires four 1-min passes with a l-min rest period between passes. Temperature rise between passes is less than 8° , and returns to 32° during the rest period. Greater than 90% lysis is obtained using this method as assessed by microscopic evaluation. The cell homogenate is separated from the glass beads by gravity, and the cell lysate is clarified by centrifugation at 3000 g for 10 min (Beckman Model J-21B). Fermentation Scale-up Results. Parameters were developed from shake-flask trials as well as previous experience for scaling up other recombinant yeast cultures. Of primary importance in recombinant yeast production is the maintenance of adequate local oxygen transfer, that is, the unidirectional rate of mass transfer (oxygen) across the interfacial area between air bubbles and liquid as well as adequate profusion of the cell. Oxygen transfer is affected by a multitude of factors including agitation, aeration rate, vessel geometry, antifoam addition, culture viscosity, cell density and metabolic rate, temperature, and pressure. In this instance, production of a low cell density culture was desirable (target A6o0 1.6) as

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TABLE III P450 EXPRESSION LEVELS UNDER VARIOUS CONDITIONS

Constr~ct

Yeast strain

Conditions

P450IIC9/pAAH5 P4501ICI0/pAAH5 P450IICI0/pAAH5

DI2 AH22 AH22

Four l-liter shaking flasks Four l-liter shaking flasks Two 10-liter fermentors

P4501IIA4/pAAH5 P4501IIA4/pAAH5

AH22 AH22

Four l-liter shaking flasks 80-liter fermentor

Yield P450/liter culture - 1 nmol - 1 . 5 nmol 2.25 nmol (single experiment) 7-11 nmol 2 nmol

P450 molecules/cell - 7 x 104 - 1 . 3 x 105 1.7 × 10~ - 8 x 105 1.5 × 105

older yeast cells tend to be difficult to lyse, and multiple generations of cells tend to encourage genetic drift or plasmid loss. Antifoam addition is maintained at a minimum, and probably does not contribute significantly to mass transfer inhibition. Adequate yeast cell profusion is conservatively considered as maintaining at least 70% dissolved oxygen concentration. Using the parameters selected in our 110-liter system, we were able to maintain no less than 90% dissolved oxygen throughout the culture period. Other factors which generally affect productivity/expression levels are inoculum level and age, specific growth rate (doubling time), pH, carbon source, nitrogen source, and sugar concentration. Optimization of pH control range and glucose feeding could well increase productivity without negatively affecting recovery and purification.

Efficiency of Expression This laboratory has used the same vector pAAH5 and the same yeast strains (D12, AH22) for two constructs, with the P450 yield varying considerably for the two constructs. Both constructs have given favorable yields in large-scale cultures (Table III).