IntroductionMammalian cytochrome P450 (P450) (Nelson et al.,1993) plays an important role in the metabolism of avariety of compounds. Saccharomyces cerevisiae was usedas the first host for the heterologous expression ofmammalian cytochrome P450 (Oeda et al., 1985).Although various P450 isozymes have been successfullyexpressed in S. cerevisiae, their expression levels fluctu-ated (Gonzalez and Korzekwa, 1995). Also, for someunknown reason, some P450 isozymes were notexpressed in S. cerevisiae (unpublished results). It hasbeen reported that the expression efficiency of P450s inS. cerevisiae can be improved by appropriate alterationof the yeast strain or the expression plasmid (Urban etal., 1990; Gonzalez and Korzekwa, 1995). However, thestructural features of cDNA inserts critical to effectiveexpression of P450s in S. cerevisiae have not been wellelucidated. Mammalian NADPH-cytochrome P450oxidoreductase (P450 reductase), an essential electrondonor to P450, has been expressed in S. cerevisiae(Ohgiya et al., 1994). The mammalian P450 reductaseshould be effectively expressed with mammalian P450sin S. cerevisiae to enhance enzymatic activity of expressedP450s. Therefore, practical methods for improving theexpression efficiency of P450s and P450 reductases inS. cerevisiae by modification of the cDNA structures haveto be developed. We report herein that trimming ofuntranslated regions (UTRs) of the cDNAs improves

the expression efficiency of the gene products in S. cere-visiae in the cases of three proteins of two P450isozymes, CYP1A1 and CYP1A2, and a protein of P450reductase.

Experimental methodsConstruction of mouse Cyp1a1 cDNA mutantsThe mouse Cyp1a1 cDNA, named MSc5, was isolatedfrom a mouse liver cDNA library (Ohgiya et al., 1991).A Hind III recognition site was introduced into the39UTR of MSc5 with an oligonucleotide MSc5-3 (59-GTGATTCAAGCTTTAGCCAG-39) according to theprotocol of the site-directed mutagenesis kit(Amersham, London). The mutated cDNA was thendigested with Hind III, and the larger moiety from the59terminus of MSc5 to the Hind III site was namedMSc5D3part. A mutant MSc5D5part containing part ofMSc5 from a Hind III site newly introduced into the59UTR to 39terminus of MSc5 was prepared by a similarmethod with another oligonucleotide MSc5-5 (59-TAGATCAAAGCTTCCATGTA-39). In the case ofMSc5D5part, the first ATG codon was removed becauseit has been reported that the second ATG codon wasthe initiation codon (Gonzalez et al., 1984; Kimura etal., 1984). A mutant MSc5D5whole3part containingpart of MSc5 from the first ATG codon to the Hind IIIsite that was the 39terminus of MSc5D3part wasprepared from MSc5D3part as follows: The DNA

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Biotechnology Letters, Vol 19, No 5, May 1997, pp. 437–441

© 1997 Chapman & Hall Biotechnology Letters · Vol 19 · No 5 · 1997 437

Improvement in the expressionefÞciency of mammalian cytochromeP450 and NADPH-cytochrome P450 oxidoreductase in Saccharomyces cerevisiaeSatoru Ohgiya*, Tsunahiro Kii1, Takako Goda, Tamotsu Hoshino, Kan Hamabuchi1, Hiroshi Yokota1, Akira Yuasa1 and Kozo IshizakiHokkaido National Industrial Research Institute, 2Ð17Ð2Ð1 Tsukisamu-higashi, Toyohira-ku, Sapporo 062, Japan and 1Department of Veterinary Biochemistry, School of Veterinary Medicine, 582Ð1 Bunkyodaimidorimachi,Ebetsu 069, Japan

Mammalian cytochrome P450 (P450) cDNAs were modiÞed by partial or complete removal of their untranslatedregions (UTRs). Expression efÞciency of P450s in Saccharomyces cerevisiae was increased by the complete removalof the UTRs from the P450 cDNAs prior to insertion into an expression vector. A similar modiÞcation was effectivein improving the expression of mammalian NADPH-P450 oxidoreductases in S. cerevisiae.

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encoding the first 47 amino acids of mouse CYP1A1was amplified from MSc5D3part by polymerase chainreaction (PCR) with Pfu DNA polymerase (Stratagene,La Jolla, USA) and a pair of primers, MSc5-Mf (59-AAGGTACCAAGCTTATGCCTTCCATGTATGG-39)and MSc5-MDr (59-AAGGTACCGGGCCCTGGTG-GAGTTCT-39). The amplified DNA was digested withHind III and Apa I. A mutant MSc5D5whole3part wasconstructed by ligation of the amplified cDNA with the39fragment of MSc5D3part digested with Apa I andHind III. A mutant MSc5Dwhole was prepared byamplification of DNA containing the coding region ofMSc5 from the first ATG codon to the terminationcodon with a pair of primers, MSc5-Mf and MSc5-Mr(59-GCAAGCTTCTAAGCCTGAAGATGCTG-39), byPCR.

Construction of mutants of mouse Cyp1a2cDNA, hamster CYP1A2 cDNA and hamsterP450 reductase cDNAThe mouse Cyp1a2 cDNA (Kimura et al., 1984) andthe hamster CYP1A2 cDNA (Lai and Chiang, 1990)were isolated from their liver cDNA libraries, andnamed MSd1 and HSd1, respectively. The nucleotidesequences of MSd1 and HSd1 in their coding regionswere identical with the corresponding reportedsequences. The hamster P450 reductase cDNA, namedHSr1, was isolated from the hamster liver cDNA library(the accession number in the DNA database: D83230;submitted for publication). The cDNAs were digestedwith Dra I to remove a part of their 39UTRs (Dpart).The other mutants were prepared by amplification ofDNAs containing their coding regions from the initia-tion codon to the termination codon (Dwhole) by PCRwith each pair of primers: MSd1f (59-TTACTAGTA-TGGCGTTCTCCCAGTAC-39) and MSd1r (59-GTAC-TAGTTCACTTGGAAAAGCGTGG-39) for MSd1;HSd1f (59-AAAAGCTTATGGCACTGTCCCAGTAC-39) and HSd1r (59-GTAAGCTTTCATTTGGAAAAA-CGTGGC-39) for HSd1; and HSr1f (59-TAAAGC-TTATGACTGAGGCTGTAGCAG-39) and HSr1r (59-GCAAGCTTCTAGCTCCACACGTCCAGG-39) forHSr1.

Expression of P450s and P450 reductases inS. cerevisiae with the cDNA mutantsThe constructed cDNAs were digested with Hind IIIfollowed by insertion into the yeast expression vectorpAAH5 carrying an alcohol dehydrogenase promoterand terminator (Ammerer, 1983). In the cases of MSc5,MSc5D3part, MSc5D5part, MSd1Dpart, HSd1Dpartand HSr1Dpart, they were blunted with T4 DNA po-lymerase and then ligated with a Hind III linker prior

to digestion with Hind III. The proper orientation ofthe insert in pAAH5 was confirmed by restrictionendonuclease mapping. S. cerevisiae AH22 (a, leu2–3,leu2–112, his4–519, can1, cir+) or YPH500 (a, ura3–52,lys2–801, ade2–101, trp1D63, his3D200, leu2D1) wastransformed with the resultant expression plasmids bythe method of Ito et al. (1983). Yeast microsomes forWestern blot analysis were prepared as follows: yeastcells at the mid-growth phase were ruptured by a Frenchpressure cell press at cell pressure of 100 atm and thencentrifuged at 9,000 g for 20 min. The 9,000 g super-natant was subsequently centrifuged at 110,000 g for60 min. The microsomal pellet was suspended in water.Yeast microsomes for determination of cytochrome creductase activity were prepared essentially according tothe method of Oeda et al. (1985). Yeast RNA wasisolated by the standard method (Ausbel et al., 1990).

Other methodsProtein concentration was determined by the Lowrymethod. Northern blot analysis was carried outaccording to the standard protocols (Ausbel et al., 1990).Radioactivity of the DNA-RNA hybrid on nitrocellu-lose filters was determined by a Molecular Imager GS-250 (Bio-Rad, Hercules, USA). P450 content in yeastmicrosomes was determined by Western blot analysiswith the anti-rat CYP1A1 antibody (Daiichi Chemical,Tokyo, Japan) according to the protocol of the ECLWestern blotting detection system (Amersham).Chemiluminescence emitted from immune complexeswas determined by the Molecular Imager GS-250. P450reductase activity was determined with cytochrome caccording to the method of Phillips and Langdon(1962).

ResultsExpression of mouse CYP1A1 in yeasttransformed with its cDNA mutants containing different lengths of the UTRsThe MSc5 mutants were prepared by partial or completeremoval of the UTRs from MSc5 (Fig. 1). The lengthsof the UTRs of the mutants are listed in Table 1. Theexpression levels of mouse CYP1A1 protein in yeastmicrosomes and mouse CYP1A1 mRNA in yeast RNAfluctuated among the yeasts transformed with themutants (Table 1). The expression levels of the proteinand mRNA in the yeast transformed with MSc5D3part(the MSc5D3part-transformant) were approximately 1.6times those in the MSc5-transformant. Moreover, theexpression levels of the mouse CYP1A1 protein in theMSc5D5whole3part-transformant increased to morethan twice that in the MSc5D3part-transformant, whilethe expression levels of the mRNA in yeast RNA were

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comparable. The expression of mouse CYP1A1 in yeastreached the highest level when MSc5Dwhole was used. On the other hand, the expression levels of theprotein and mRNA in the MSc5D5part-transformantwere lower than those in the MSc5-transformant.

Expression of mouse CYP1A2, hamsterCYP1A2 and hamster P450 reductase in yeasttransformed with their cDNA mutantsWe examined whether or not the complete removal ofUTRs from the Cdnas improved the expression effi-ciency of the corresponding proteins in cases of otherP450s and P450 reductases. The mutants of mouse

Cyp1a2 cDNA, hamster CYP1A2 cDNA, and hamsterP450 reductase cDNA containing part of their UTRs(Dpart) or no UTRs (Dwhole) were prepared for expres-sion of the gene products in S. cerevisiae. The expressionlevels of mouse and hamster CYP1A2 proteins werehigher in the yeast transformed with the Dwholemutants than those in the yeast transformed with theDpart mutants (Table 2). Surprisingly, mouse CYP1A2was successfully expressed in the MSd1Dwhole-trans-formant but only faintly expressed in the MSd1Dpart-transformant (Fig. 2). Hamster P450 reductase wasfunctionally expressed in the HSr1Dpart-transformant,judging from the increased activity of cytochrome creductase of the yeast microsomes (Table 3). The expres-sion level of hamster P450 reductase in the microsomes

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Biotechnology Letters · Vol 19 · No 5 · 1997 439

Table 1 Comparison of expression levels of mouseCYP1A1 protein and its mRNA in yeast transformedwith MSc5 mutants containing various lengths of UTRs

Relative contents Length of UTRof CYP1A1

Protein* mRNA* 59 39

MSc5 100 100 73 947MSc5D3part 159 157 73 48MSc5D5whole3part 348 144 0 48MSc5Dwhole 374 184 0 0MSc5D5part 22 28 2 947

* Values are the means for samples prepared from three indepen-dent transformants. Expression levels of mouse CYP1A1 proteinand mRNA in yeast transformed with MSc5 are deÞned as 100.

Figure 1 Structure of mouse Cyp1a1 cDNA mutants.Nucleotide sequences around the initiation and terminationcodons are displayed. The initiation and termination codonsare underlined. The UTRs are displayed with particalnucleotide sequences in small letters and number ofnucleotides.

Table 2 Comparison of expression levels of CYP1A2proteins in yeast transformed with mouse and hamsterCyp1a2 cDNA mutants containing various lengths ofUTRs

Relative content Length of UTRof CYP1A2 protein

59 39

MSd1Dpart 100 51 55MSd1Dwhole 1290 0 0HSd1Dpart 100 10 69HSd1Dwhole 140 0 0

* Values are the means for samples prepared from three indepen-dent transformants. The expression level of CYP1A2 protein in yeasttransformed with each Dpart mutant is deÞned as 100.

Table 3 Comparison of P450 reductase activities in microsomes of yeast transformed with P450 reductase cDNAmutants

cDNA P450 reductase activity* (nmol Length of UTR Referencecytochrome c reduced/min/mg protein)

59 39

None** 32.6HSr1Dpart 98.5 62 335HSr1Dwhole 569 0 0dR25Dpart 28.6 27 309 Ohgiya et al., 1994dR25Dwhole 279 0 0 Ohgiya et al., 1994

* Values are the means for samples prepared from three independent transformants.** The activity of yeast P450 reductase was determined as background.

Figure 2 Western blot analysis of mouse CYP1A2 inmicrosomes of yeast transformed with MSd1 mutants. Eachlane contains 8mg of microsomal proteins. Lanes 1Ð3,MSd1Dpart tansformants; lane 4Ð6, MSd1D whole transfor-mants.

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of the HSr1Dwhole-transformant was much higher thanthat in the microsomes of the HSr1Dpart-transformant,implying that the complete removal of the UTRs fromthe hamster P450 reductase cDNA was effective inincreasing the gene product in S. cerevisiae (Table 3).We have reported a similar result in the case of themouse P450 reductase cDNA, dR25 (Table 3; Ohgiyaet al., 1994).

DiscussionThe effects of the removal of the UTRs from the mouseCyp1a1 cDNA, MSc5, on transcription and translationlevels of mouse CYP1A1 were first examined. Thepartial removal of the 39UTR from MSc5 improved the expression efficiency of the protein in S. cerevisiae.The mRNA increased as much as the protein in yeast.In addition, the removal of the remaining 39UTR from MSc5DD5whole3part increased the mRNA level as much as the protein level (Table 1;MSc5DD5whole3part and MSc5Dwhole). These resultsindicate that the increase of the protein is regulated inthe pretranslational phase. The pretranslational mecha-nism of increasing the mouse CYP1A1 mRNA isunclear at present. Shaw and Kamen (1986) reportedthat the AUUUA sequence in 39UTR of mRNAs accel-erated degradation of the mRNA in mammalian cells.The mouse Cyp1a1 cDNA has the AUUUA sequencein the 39UTR. In addition, the transferrin receptormRNA and the thyrotropin-releasing hormone receptormRNA were also reported to contain sequences respon-sible for instability of their mRNAs in their 39UTRs(Klausner and Harford, 1989; Narayanan et al., 1992).Accordingly, the removal of the 39UTRs from MSc5might stabilize the mRNA itself by loss of a sequenceresponsible for degradation of the mRNA.

The removal of the 59UTR from MSc5 together withthe first ATG codon reduced the expression levels ofthe mRNA and the protein, implying that the struc-ture of the 59UTR of the mRNA also affected theexpression efficiency of the protein in the pretransla-tional phase (Table 1; MSc5 and MSc5D5part). On theother hand, the complete removal of the 59UTR fromMSc5D3part enhanced its expression efficiency of thegene product in yeast (Table 1; MSc5D3part andMSc5DD5whole3part). Since the mRNA levels aresimilar, the increase in the protein level is thought tobe due to an increase in the translational efficiency.Cullin and Pompon (1988) have pointed out that thedistance between the 59-terminus and the initiationcodon is inversely correlated with the translational effi-ciency of the mRNA. Pompon (1988) has also reportedthat the expression efficiency of mouse CYP1A1

changed with changes in the 59-terminal position of itscDNA. Our results suggest that not only the length ofthe 59UTR but also the nucleotide sequence of the59UTR are correlated with translational efficiency aswell as pretranslational efficiency in the expression ofmouse CYP1A1.

The mouse CYP1A1 protein expressed in the yeasttransformed with MSc5Dwhole, the UTR-less cDNA,was 3 times greater than that expressed in the yeasttransformed with the original cDNA. Accordingly, thecomplete removal of the UTRs from the mouseCYP1A1 cDNA was thought to be effective inimproving the expression efficiency of mouse CYP1A1in S. cerevisiae. To examine whether this strategy couldbe applied to the expression of other P450 isozymes andP450 reductase, mouse CYP1A2, hamster CYP1A2 andhamster P450 reductase were expressed in yeast trans-formed with their cDNA mutants. The proteins wereexpressed in the yeast transformed with the corre-sponding UTR-less cDNA mutants more than in theyeast transformed the cDNA mutants containing partof the UTRs (Tables 2 and 3). Especially, in the casesof mouse CYP1A2 and mouse P450 reductase (Ohgiyaet al., 1994), the proteins were successfully expressed inthe yeast transformed with the UTR-less cDNA mutantwhile they were not expressed in the yeast transformedwith the cDNA mutant containing part of the UTRs(Tables 2 and 3). Thus, the existence of the UTRs ofP450 and P450 reductase cDNAs may be critical, insome cases, to the expression efficiency of their geneproducts in S. cerevisiae.

The guinea pig P450 reductase (Ohgiya et al., 1992)was not expressed in S. cerevisiae transformed with itscDNA mutant containing part of the UTRs (data notshown). However, the enzyme was functionallyexpressed in CHL cell lines transformed with the samecDNA (Sawada et al., 1993). This result implies thatthe effect of the UTRs of the cDNAs on expression effi-ciency of the gene products in mammalian cells isdifferent from that in yeast.

On these grounds, we consider that the completeremoval of the UTRs from P450 and P450 reductasecDNAs is effective in improving their expression effi-ciency in S. cerevisiae.

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Received 5 February 1997; Revisions requested 14 February 1997;

Revisions received 17 March 1997; Accepted 21 March 1997

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