Modified P450 reductase with N-terminal deletions and endoplasmic reticulum retention tag

Abstract

The present invention provides an isolated or recombinant polypeptide comprising or consisting of a modified P450 reductase which lacks N-terminal amino acids relative to the corresponding wild type P450 reductase and comprises an epitope tag comprising the sequence HDEL or KDEL. The modified P450 reductase, when co-expressed with a cytochrome P450, increases the activity and/or expression of the cytochrome P450 compared to the activity and/or expression of the cytochrome P450 when co-expressed with the wild type P450 reductase.

Claims

1. An isolated or recombinant polypeptide comprising or consisting of a modified P450 reductase, wherein the modified P450 reductase is a P450 reductase which lacks N-terminal amino acids relative to the corresponding wild type P450 reductase and comprises an epitope tag comprising the amino acid sequence HDEL (SEQ ID NO: 1), optionally wherein the amino acid sequence HDEL (SEQ ID NO: 1) is replaced by KDEL (SEQ ID NO: 2), and wherein the modified P450 reductase has the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4, or an amino acid sequence which has at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4, and further wherein the modified P450 reductase, when co-expressed with a cytochrome P450, increases the activity and/or expression of the cytochrome P450 compared to the activity and/or expression of the cytochrome P450 when co-expressed with the wild type P450 reductase.

2. The polypeptide as claimed in claim 1, wherein the P450 reductase lacks N-terminal amino acids by being truncated at the N terminus.

3. The polypeptide as claimed in claim 2, wherein the truncation comprises the 24 N-terminal amino acids.

4. The polypeptide as claimed in claim 3, wherein the truncation comprises the 54 N-terminal amino acid acids.

5. The polypeptide as claimed in claim 1, wherein the epitope tag is linked to the C-terminal end of the polypeptide.

6. The polypeptide of claim 1, wherein the P450 reductase is a human P450 reductase.

7. The polypeptide of claim 1 wherein the P450 reductase is a yeast P450 reductase.

8. The polypeptide of claim 1, wherein the modified P450 reductase has an amino acid sequence which has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4.

Description

EXAMPLES

(1) The present invention will now be described in more detail with reference to the following non-limiting examples. Reference is made to the accompanying drawings in which:

(2) FIG. 1 illustrates the plasmid pBluKS(+)/ADH2p-573.

(3) FIG. 2 illustrates the plasmid pSYE263.

(4) FIG. 3 illustrates the plasmid pSYE264.

(5) FIG. 4 illustrates the plasmid pSYE265.

(6) FIG. 5 illustrates the basic yeast integrating plasmid YILEUGAL1S.

(7) FIG. 6 illustrates the plasmid YILEUADH2S.

(8) FIG. 7 is a comparison of the protein sequences of wt-hRD (SEQ ID NO: 6), N1hRD (SEQ ID NO: 7) and N2hRD (SEQ ID NO: 8).

(9) FIG. 8 is a comparison of the protein sequences of wt-hRD (SEQ ID NO: 6), N1hRD-M (SEQ ID NO: 9), N1hRD-HDEL (SEQ ID NO: 3) and N2hRD-HDEL (SEQ ID NO: 4).

(10) FIG. 9 shows the nucleic acid sequence of the BamHI-XbaI DNA fragment of the N1hRD-M gene chemically synthesized using yeast biased codons (SEQ ID NO: 5); the Start Site is at base 12 and indicated in bold.

(11) FIG. 10 illustrates the integrating plasmid, YILEUADH2S, that bears the N1hRD-M gene.

(12) FIG. 11 illustrates the integrating plasmid that bears the N1hRD-HDEL gene in YILEUADH2S.

(13) FIG. 12 illustrates the integrating plasmid that bears the N2hRD-HDEL gene in YILEUADH2S.

(14) FIG. 13 is a graph comparing ROS produced by the human N1hRD-M gene (isolated from a human liver cDNA library) and the one synthesized using yeast-biased codons.

(15) FIG. 14 is a graph comparing of ROS produced by three hRD variant genes, synthesized using yeast-biased codons.

EXAMPLE 1CLONING OF THE 573 BP YEAST ADH2 PROMOTER AS A SALI(NGOMIV)-(HINDIII)BAMHI FRAGMENT IN PBLUESCRIPT

(16) The cloning of a SalI(NgoMIV)-(HindIII)BamHI ADH2 promoter fragment (SEQ ID 1) in pBlueScriptII SK(+) was performed using ADH2 promoter sequence specific primers (5 PCR primer: 5-CCGGTCGACG CCGGCGGCAA AACGTAGGGG CAAACAAACG G-3 (SEQ ID NO: 10the first six letters in italics signify the SalI site and the next six letters represent the NgoMIV site) & 3 PCR primer: 5-CGGGATCCAA GCTTTGTGTA TTACGATATA GTTAATAG-3 (SEQ ID NO: 11the first six letters in italics signify the BamHI site and the next six letters represent the HindIII site). The amplified fragment, digested with SalI-BamHI, was cloned in pBlueScriptII KS(+) digested with SalI-BamHI.

(17) TABLE-US-00001 TheADH2promoter. (SEQIDNO:12) 1CCGGTCGACGCCGGCGGCAAAACGTAGGGGCAAACAAACG GAAAAATCGT 51TTCTCAAATTTTCTGATGCCAAGAACTCTAACCAGTCTTA TCTAAAAATT 101GCCTTATGATCCGTCTCTCCGGTTACAGCCTGTGTAACTG ATTAATCCTG 151CCTTTCTAATCACCATTCTAATGTTTTAATTAAGGGATTT TGTCTTCATT 201AACGGCTTTCGCTCATAAAAATGTTATGACGTTTTGCCCG CAGGCGGGAA 251ACCATCCACTTCACGAGACTGATCTCCTCTGCCGGAACAC CGGGCATCTC 301CAACTTATAAGTTGGAGAAATAAGAGAATTTCAGATTGAG AGAATGAAAA 351AAAAAAAAAAAAAAAAGGCAGAGGAGAGCATAGAAATGGG GTTCACTTTT 401TGGTAAAGCTATAGCATGCCTATCACATATAAATAGAGTG CCAGTAGCGA 451CTTTTTTCACACTCGAAATACTCTTACTACTGCTCTCTTG TTGTTTTTAT 501CACTTCTTGTTTCTTCTTGGTAAATAGAATATCAAGCTAC AAAAAGCATA 551CAATCAACTATCAACTATTAACTATATCGTAATACACAAA GCTTGGATCC 601CG

(18) One correct clone obtained after ligation and transformation in DH5alpha bacterial cells was named pBluKS(+)/ADH2p-573 (FIG. 1) and was used for further cloning in a 2-micron and an integrating yeast expression vector. The veracity of the clone was confirmed by restriction enzyme analysis and corroborated by DNA sequencing.

EXAMPLE 2CLONING OF THE ADH2 PROMOTER IN A YEAST 2-MICRON VECTOR

(19) A 585 bp NgoMIV-BamHI ADH2 promoter fragment was isolated from pBluKS(+)/ADH2p-573 (FIG. 1) and cloned in pYES2 (a yeast 2-micron vector obtained from Invitrogen) digested with NgoMIV and BamHI to obtain the plasmid pSYE263 (FIG. 2).

EXAMPLE 3CLONING OF THE HUMAN CYP2D6 GENE IN PSYE263

(20) A 1506 bp BamHI-XbaI fragment containing the human CYP2D6 gene (SEQ ID No. 2) was cloned in pSYE263 (FIG. 2), digested with BamHI and XbaI, to obtain the plasmid pSYE264 (FIG. 3).

(21) TABLE-US-00002 ThehumanCYP2D6gene(1512bp)asclonedfrom ahumanlivercDNAlibrary(SEQIDNO:13): 1GGATCCAAAAAAATGGGGCTAGAAGCACTGGTGCCCCTGG CCGTGATAGT 51GGCCATCTTCCTGCTCCTGGTGGACCTGATGCACCGGCGC CAACGCTGGG 101CTGCACGCTACCCACCAGGCCCCCTGCCACTGCCCGGGCT GGGCAACCTG 151CTGCATGTGGACTTCCAGAACACACCATACTGCTTCGACC AGTTGCGGCG 201CCGCTTCGGGGACGTGTTCAGCCTGCAGCTGGCCTGGACG CCGGTGGTCG 251TGCTCAATGGGCTGGCGGCCGTGCGCGAGGCGCTGGTGAC CCACGGCGAG 301GACACCGCCGACCGCCCGCCTGTGCCCATCACCCAGATCC TGGGTTTCGG 351GCCGCGTTCCCAAGGGGTGTTCCTGGCGCGCTATGGGCCC GCGTGGCGCG 401AGCAGAGGCGCTTCTCCGTGTCCACCTTGCGCAACTTGGG CCTGGGCAAG 451AAGTCGCTGGAGCAGTGGGTGACCGAGGAGGCCGCCTGCC TTTGTGCCGC 501CTTCGCCAACCACTCCGGACGCCCCTTTCGCCCCAACGGT CTCTTGGACA 551AAGCCGTGAGCAACGTGATCGCCTCCCTCACCTGCGGGCG CCGCTTCGAG 601TACGACGACCCTCGCTTCCTCAGGCTGCTGGACCTAGCTC AGGAGGGACT 651GAAGGAGGAGTCGGGCTTTCTGCGCGAGGTGCTGAATGCT GTCCCCGTCC 701TCCTGCATATCCCAGCGCTGGCTGGCAAGGTCCTACGCTT CCAAAAGGCT 751TTCCTGACCCAGCTGGATGAGCTGCTAACTGAGCACAGGA TGACCTGGGA 801CCCAGCCCAGCCCCCCCGAGACCTGACTGAGGCCTTCCTG GCAGAGATGG 851AGAAGGCCAAGGGGAACCCTGAGAGCAGCTTCAATGATGA GAACCTGCGC 901ATAGTGGTGGCTGACCTGTTCTCTGCCGGGATGGTGACCA CCTCGACCAC 951GCTGGCCTGGGGCCTCCTGCTCATGATCCTACATCCGGAT GTGCAGCGCC 1001GTGTCCAACAGGAGATCGACGACGTGATAGGGCAGGTGCG GCGACCAGAG 1051ATGGGTGACCAGGCTCACATGCCCTACACCACTGCCGTGA TTCATGAGGT 1101GCAGCGCTTTGGGGACATCGTCCCCCTGGGTATGACCCAT ATGACATCCC 1151GTGACATCGAAGTACAGGGCTTCCGCATCCCTAAGGGAAC GACACTCATC 1201ACCAACCTGTCATCGGTGCTGAAGGATGAGGCCGTCTGGG AGAAGCCCTT 1251CCGCTTCCACCCCGAACACTTCCTGGATGCCCAGGGCCAC TTTGTGAAGC 1301CGGAGGCCTTCCTGCCTTTCTCAGCAGGCCGCCGTGCATG CCTCGGGGAG 1351CCCCTGGCCCGCATGGAGCTCTTCCTCTTCTTCACCTCCC TGCTGCAGCA 1401CTTCAGCTTCTCGGTGCCCACTGGACAGCCCCGGCCCAGC CACCATGGTG 1451TCTTTGCTTTCCTGGTGAGCCCATCCCCCTATGAGCTTTG TGCTGTGCCC 1501CGCTAGTCTAGA

EXAMPLE 4CLONING OF THE HUMAN CYP1A2 GENE IN PSYE263

(22) A 1563 bp BamHI-XhoI fragment containing the human CYP1A2 gene (SEQ ID No. 12) was cloned in pSYE263 (FIG. 2), digested with BamHI and XhoI, to obtain the plasmid pSYE265 (FIG. 4).

(23) TABLE-US-00003 ThehumanCYP1A2gene(1573bp)asclonedfroma humanlivercDNAlibrary(SEQIDNO:14). 1ATGGATCCAAAAAAATGGCATTGTCCCAGTCTGTTCCCTT CTCGGCCACA 51GAGCTTCTCCTGGCCTCTGCCATCTTCTGCCTGGTATTCT GGGTGCTCAA 101GGGTTTGAGGCCTCGGGTCCCCAAAGGCCTGAAAAGTCCA CCAGAGCCAT 151GGGGCTGGCCCTTGCTCGGGCATGTGCTGACCCTGGGGAA GAACCCGCAC 201CTGGCACTGTCAAGGATGAGCCAGCGCTACGGGGACGTCC TGCAGATCCG 251CATTGGCTCCACGCCCGTGCTGGTGCTGAGCCGCCTGGAC ACCATCCGGC 301AGGCCCTGGTGCGGCAGGGCGACGATTTCAAGGGCCGGCC TGACCTCTAC 351ACCTCCACCCTCATCACTGATGGCCAGAGCTTGACCTTCA GCACAGACTC 401TGGACCGGTGTGGGCTGCCCGCCGGCGCCTGGCCCAGAAT GCCCTCAACA 451CCTTCTCCATCGCCTCTGACCCAGCTTCCTCATCCTCCTG CTACCTGGAG 501GAGCATGTGAGCAAGGAGGCTAAGGCCCTGATCAGCAGGT TGCAGGAGCT 551GATGGCAGGGCCTGGGCACTTCGACCCTTACAATCAGGTG GTGGTGTCAG 601TGGCCAACGTCATTGGTGCCATGTGCTTCGGACAGCACTT CCCTGAGAGT 651AGCGATGAGATGCTCAGCCTCGTGAAGAACACTCATGAGT TCGTGGAGAC 701TGCCTCCTCCGGGAACCCCCTGGACTTCTTCCCCATCCTT CGCTACCTGC 751CTAACCCTGCCCTGCAGAGGTTCAAGGCCTTCAACCAGAG GTTCCTGTGG 801TTCCTGCAGAAAACAGTCCAGGAGCACTATCAGGACTTTG ACAAGAACAG 851TGTCCGGGACATCACGGGTGCCCTGTTCAAGCACAGCAAG AAGGGGCCTA 901GAGCCAGCGGCAACCTCATCCCACAGGAGAAGATTGTCAA CCTTGTCAAT 951GACATCTTTGGAGCAGGATTTGACACAGTCACCACAGCCA TCTCCTGGAG 1001CCTCATGTACCTTGTGACCAAGCCTGAGATACAGAGGAAG ATCCAGAAGG 1051AGCTGGACACTGTGATTGGCAGGGAGCGGCGGCCCCGGCT CTCTGACAGA 1101CCCCAGCTGCCCTACTTGGAGGCCTTCATCCTGGAGACCT TCCGACACTC 1151CTCCTTCTTGCCCTTCACCATCCCCCACAGCACAACAAGG GACACAACGC 1201TGAATGGCTTCTACATCCCCAAGAAATGCTGTGTCTTCGT AAACCAGTGG 1251CAGGTCAACCATGACCCAGAGCTGTGGGAGGACCCCTCTG AGTTCCGGCC 1301TGAGCGGTTCCTCACCGCCGATGGCACTGCCATTAACAAG CCCTTGAGTG 1351AGAAGATGATGCTGTTTGGCATGGGCAAGCGCCGGTGTAT CGGGGAAGTC 1401CTGGCCAAGTGGGAGATCTTCCTCTTCCTGGCCATCCTGC TACAGCAACT 1451GGAGTTCAGCGTGCCGCCGGGCGTGAAAGTCGACCTGACC CCCATCTACG 1501GGCTGACCATGAAGCACGCCCGCTGTGAACATGTCCAGGC GCGGCTGCGC 1551TTCTCCATCAACTGACTCGAGAT

EXAMPLE 5CLONING OF THE ADH2 PROMOTER IN A YEAST INTEGRATING VECTOR

(24) A 591 bp SalI-BamHI ADH2 promoter fragment was isolated from pBluKS(+)/ADH2p-573 (FIG. 1) and cloned in YILEUGAL1 S (FIG. 5; a yeast LEU2-integrating vector created in-house) digested with SalI and BamHI to obtain the plasmid YILEUADH2S (FIG. 6). S signifies the SUC2 gene terminator.

EXAMPLE 6CONSTRUCTION OF THE HRD VARIANT GENES

(25) Different variants of the hRD gene were constructed to obtain hRD activity that may not be deleterious for P450 expression. The aim was to devise an optimal system that allows better production of human P450 isozymes in yeast. The ultimate goal was to find an alternative system for the production of recombinant human P450 isozymes not only in yeast but also in insect and mammalian cells.

(26) For yeast expression, the first variant gene of the human P450 reductase was constructed via chemical synthesis of the gene using yeast-biased codons. The other two variants were constructed from the first via PCR using sequence specific primers.

(27) The first variant lacks the negatively charged (5 negatively charged amino acids+a potential positively charged amino acid) N-terminal 24 amino acids and the COOH-terminal Stop codon (FIG. 7; N1hRDSEQ ID NO: 7) but contains the c-myc epitope tag EQKLISEEDLNG (SEQ ID NO: 15) at the COOH-terminal end (FIG. 8; N1hRD-MSEQ ID NO: 9). The 12 amino acid c-myc tag is also a negatively charged peptide (containing 4 negatively charged amino acids and a positively charged amino acid) and is linked to the C-terminus of hRD. The c-myc tag also allows monitoring of P450 reductase protein production inside the cell. This variant is referred to as N1hRD-M (SEQ ID NO: 9).

(28) The second variant also lacks the negatively charged N-terminal 24 amino acids and the COOH-terminal Stop codon (FIG. 7; N1hRDSEQ ID NO: 7) but contains a 4-amino acid yeast endoplasmic reticular retention signal, HDEL (SEQ ID NO: 1) (FIG. 8; N1hRD-HDELSEQ ID NO: 3). The DNA sequence was constructed via using a 3-primer that codes for the HDEL sequence. This variant has been named N1hRD-HDEL.

(29) The third variant lacks the 54-amino acid membrane anchoring region of human P450 reductase (hRD) and the COOH-terminal Stop codon (FIG. 7) but contains a 4-amino acid yeast endoplasmic reticular retention signal, HDEL (SEQ ID NO: 2) (FIG. 8; N2hRD-HDELSEQ ID NO: 4). The DNA sequence was constructed via PCR using a 5-primer that allows deletion of another 30 amino acids from that 3-primer that codes for the HDEL sequence. This variant has been named N2hRD-HDEL.

EXAMPLE 7CONSTRUCTION OF YEAST INTEGRATING PLASMIDS THAT BEAR VARIANTS OF THE HUMAN P450 REDUCTASE (HRD) GENE UNDER THE CONTROL OF THE 573 BP ADH2 PROMOTER

(30) The hRD variants and the full-length hRD gene were cloned in the yeast integrating vector that would allow expression of hRD under the control of the ADH2 promoter (YILEUADH2MS; FIG. 6). The steps involved were: (1) Cloning of the hRD variants from a human liver cDNA library in pBlueScript vectors and confirming the inserts via restriction enzyme analysis and DNA sequencing. (2) Sub-cloning the hRD variant gene, N1hRD-M, chemically synthesized using yeast biased codons and confirmed by DNA sequencing FIG. 9), in a yeast integrating vector that contains the 573 bp ADH2 promoter-SUCt (ADH2-573 promoter+SUC2t) cassette. The hRD gene variant is cloned downstream of the ADH2-573 promoter and upstream of the SUC2 terminator to obtain the plasmid YILEUADH2S/N1hRD-M (FIG. 10). (3) Constructing via PCR and unique primers the hRD variant genes, N1hRD-HDEL and N2hRD-HDEL, from the N1hRD-M clone which had been synthesised using yeast biased codons. (4) Sub-cloning the hRD variant genes, N1hRD-HDEL and N2hRD-HDEL, using yeast biased codons, in a yeast integrating vector that contains the 573 bp ADH2 promoter-SUCt (ADH2-573 promoter+SUC2t) cassette. The hRD gene variants are cloned downstream of the ADH2-573 promoter and upstream of the SUC2 terminator to obtain the plasmids YILEUADH2S/N1hRD-HDEL and YILEUADH2S/N2hRD-HDEL (FIGS. 11 and 12).

EXAMPLE 8EXPRESSION OF HUMAN CYP GENES IN YEAST

(31) The hRD variant genes, (N1hRD-M, N1hRD-HDEL, N2hRD-HDEL), were integrated into Saccharomyces cerevisiae strain W303B (MAT a leu2 his3 trp1 can1-100 ade2 trp1 ura3) using standard yeast transformation procedures as detailed below. The integrating plasmids, bearing the N1hRD-M, N1hRD-HDEL, N2hRD-HDEL genes, were first linearised with the restriction enzyme BstEII before introducing linearised DNA into yeast cells via homologous recombination. The resultant strains were named: a) W303B-N1hRD-M, b) W303B-N1hRD-HDEL, c) W303B-N2hRD-HDEL.

(32) They were used for the transformation of the yeast episomal plasmids that bear the CYP2D6 and CYP1A2 genes: 1. pSYE264 (bearing the human CYP2D6 gene), 2. pSYE265 (bearing the human CYP1A2 gene).

(33) Yeast Transformation

(34) A single colony from the strains W303B-N1hRD-M, W303B-N1hRD-HDEL, W303B-N2hRD-HDEL were picked up from a minimal medium (SD) plate (supplemented with appropriate nutrients depending on the auxotrophic markers in the yeast strain) and inoculated into 10 ml of YPD medium (2% Bacto Peptone, 1% yeast extract, 2% glucose). The cells were grown overnight at 30 C. with 220 rpm shaking. 1.5 ml of overnight cultures were centrifuged at 13,000 rpm for a few seconds to collect the cell pellets. 0.5-2 g of transforming DNA (i.e. the CYP bearing expression plasmids, pSYE264 and pSYE265) and 100 g of single-stranded salmon sperm DNA were added to pellets and vortexed briefly. 500 l of PEG solution (40% PEG 3350, 0.1M lithium acetate pH 7.5, 10 mM Tris-HCl pH 7.5, 1 mM EDTA pH7.5) and 5-10% DMSO were added to transformation mixes. All mixes were incubated in a Thermo-mixer for 15 min at 25 C. with shaking at 400 rpm, and then were heat shocked for 15 min at 42 C. After 10 min, 5-10% ethanol was added. The cells were pelleted at 8000 rpm for 1 min and were washed twice in 1TE buffer and re-suspended in 250 l-500 l 1TE pH7.5. The cells were plated out on SD agar medium and incubated at 30 C. for 2-3 days.

(35) The transformants were named: 1. W303B-N1hRD-M:pSYE264, 2. W303B-N1hRD-M:pSYE265, 3. W303B-N1hRD-HDEL:pSYE264, 4. W303B-N1hRD-HDEL:pSYE265, 5. W303B-N2hRD-HDEL:pSYE264, 6. W303B-N2hRD-HDEL:pSYE265.

(36) Yeast Cultures for Microsome Preparation

(37) Recombinant yeast cells were grown in culture using the following protocol: 1. On day one, a loopful of fresh yeast cells from an SD-agar plate was inoculated in 20 ml of SD media (1.34 g/200 ml of yeast nitrogen base) containing required nutrients, 2% glucose, and 0.02% casein enzymatic hydrolysate (casamino acids, Sigma, C-7585; containing all the twenty essential amino acids). The cultures were grown overnight at 30 C. with shaking at 220 rpm. 2. On day two, once OD.sub.600 (i.e. OD measured at 600 nm) of the cultures reached 5 to 6 OD-s, the cultures were inoculated into 400 ml YPD medium (1% Bacto Peptone, 1% yeast extract, 2% glucose) with appropriate nutrients in 2-liter flask. The YPD cell culture was incubated at 30 C. at 220 rpm for 16 hours. 3. On day three, after 16 hours, optical density was again measured at 600 nm after diluting the original culture 1:10. Once an optical density of the YPD media cell culture reached between 14 and 21 OD.sub.600, the culture was kept at 4 C. 4. Day 3 continued: a centrifuge was pre-chilled to 4 C. The cell culture grown in YPD broth was transferred into a sterile bucket and was centrifuged at 3622 rpm for 15 minutes. A pellet formed at the bottom of the bucket and the supernatant was poured away. 150 ml of Harvest Buffer (118.2 g of 0.65 M Sorbitol, 10 ml of 1 M Tris-HCl, pH 7.5, 200 l of 0.5 M EDTA, pH 8.0 made up to a liter) was added; the pellet was gently re-suspended and then centrifuged at 3622 rpm for 15 minutes. At the final step, supernatant was poured away and the dry pellet was frozen at 80 C. and the pellet weight was recorded. The pellets can be kept at 80 C. for any length of time before beginning the microsome preparations.

(38) Microsome Preparation

(39) Microsome preparation is the process where the yeast cells are broken down and differentially centrifuged so that the unbroken cells, nuclei, mitochondria and other cell debris are sedimented out and the endoplasmic reticulum (ER) containing cytochrome P450s are obtained in the supernatant. Unwanted soluble matter is later separated from the ER by further centrifugation or PEG precipitation. The colour of the supernatant is reddish brown due to the presence of haeme, an iron-containing co-factor. The following steps in the procedure outline the method by which microsomes expressing a reductase (wild type or variant) or a CYP enzyme (co-expressed with a variant reductase) were obtained.

(40) The cell pellet that had been obtained from the earlier cell culture was weighed and the weight of cell pellet was recorded. The pellet was gently re-suspended in Harvest Buffer containing 100 mM dithiothreitol (DTT) and 100 mM 4-(2-aminoethyl) benzene sulphonylflouride HCl (AEBSF). 1 g of cell pellet was re-suspended in 1.4 ml of Harvest Buffer containing a general protease inhibitor (i.e. 100 ml of Harvest Buffer+0.266 ml DTT (100 mM)+2.66 ml AEBSF (100 mM)). The cell suspension was cooled to 4 C. Cells were subjected to disruption using a cell disrupter (Constant Systems), pressure was maintained at 22.5 KPSI with a single shot disrupter head. The disrupted cells were centrifuged for 15 minutes at 4500 rpm at 4 C. The volume of supernatant was multiplied by 3.75 to give the volume of DMB TES buffer (10 ml of Tris-HCl (1 M) pH 8.0, 400 l of EDTA 0.5 M pH 8.0, 30 ml of 4 M sorbitol, made up to 200 ml) that were used. This volume was divided by 40 to give the volume of NaCl, and NaCl volume was divided by 10 to give the volume of PEG solution that were used. A 50% PEG3350 solution was added drop-wise to the supernatant and then put through three high-speed centrifugations and the suspension was mixed gently. The concentrated suspension mixture was then left in the cold room on ice for 20 minutes after which it was centrifuged using the JL10 rotor at 9333 rpm for 20 minutes. The microsome pellet was obtained at the bottom of the bucket and the pellet was then washed with Harvest Buffer twice to remove the remaining 50% PEG3350 solution. Then gently, with the help of a spatula, the pellet was removed and transferred to a homogenizer tube and approximately 5 ml of DMB B buffer (1 ml of 1 M Tris-HCl pH 7.5, final concentration 10 mM, 200 l of 0.5 M EDTA pH 8.0, final concentration 1.0 mM, 40 ml 20% Glycerol, made up to 100 ml) was added. Microsomes were homogenised gently and then aliquoted in to eppendorf tubes so that the aliquots could be stored at 80 C.

(41) Determination of Total Microsomal Protein Concentrations

(42) Protein concentrations in all microsomal samples were measured using the Bio-Rad Bradford protein estimation kit. The Bradford dye (consisting of Coomassie Brilliant Blue G-250 dye) when mixed with a protein sample changes colour from brown to blue and the colour change is proportional to the amount of protein present in the sample. The intensity of the colour is then compared to the colour seen in protein solutions obtained through serial dilutions of a stock solution of a standard protein, bovine serum albumin (BSA). Each dilution of BSA has a defined protein concentration. Comparison with the BSA standard curve allows determination of the concentration of proteins present in any microsomal sample. For measurements of intensity of the blue colour, absorbance is measured at a wavelength of 595 nm using 96-well flat-bottomed microtitre plates and a Bio-Tek Synergy HT plate reader.

(43) Measurement of total protein concentrations allowed determination of the amount of P450 in a specific amount of total protein. This was essential for standardization of P450 enzymatic assays.

(44) Determination of P450 Amounts Via CO-Difference Spectra

(45) Difference spectra of microsomal preparations were measured in a dual-beam spectrophotometer (Shimadzu) using plastic disposable cuvettes. 850 l of a solution containing 100 mM potassium phosphate and 20% glycerol (pH7.5) was added to the cuvette, and left for one minute. Then a few grains of sodium hydrosulfite was added, mixed gently to prevent any bubble forming in the cuvette and left for another minute. 150 l of microsomes were added into the cuvette and the whole suspension was mixed gently. Two cuvettes (one containing sodium hydrosulphite without microsome and the other with microsome) were prepared and a baseline of light absorption of the buffer and microsome mixture was recorded in the dual-beam spectrophotometer from 400 nm to 500 nm. Carbon monoxide was bubbled slowly into one sample cuvette for about one minute, 1 bubble/second. Light absorption was recorded again from 400 nm to 500 nm. The concentration of cytochrome P450 in the cuvette was calculated from the absorption change at 450 nm relative to the absorbance change at 490 nm, using the formula below:
P450 content (nmole/ml)=(A.sub.450A.sub.490)df1000/extinction coefficient 450 nm
P450 concentration (nmole/mg protein)=P450 content/total protein
df=dilution factor(total volume in cuvutte/volume microsome)
Extinction Coefficient 420 nm=110 mM.sup.1 cm.sup.1
Extinction Coefficient 450 nm=91 mM.sup.1 cm.sup.1

(46) Extinction coefficient is the fraction of light lost to scattering and absorption per unit distance in a participating medium. It is the sum of absorption coefficient and scattering coefficient.

(47) Results

(48) The relative amounts of P450 obtained using the different P450 reductase variants (hRD, N1hRD-M, N1hRD-HDEL, N2hRD-HDEL) are shown below in Table 1 and Table 2.

(49) TABLE-US-00004 TABLE 1 The relative amounts of CYP2D6 obtained using the P450 reductase variants, hRD, N1hRD-M, N1hRD-HDEL and N2hRD-HDEL (genes synthesized using yeast biased codons). Relative Amounts of Human P450 Reductase Variant CYP2D6 Obtained hRD (SEQ ID NO: 6) 1 N1hRD-M (SEQ ID NO: 9) .sup.3 10% N1hRD-HDEL(SEQ ID NO: 3) 3.8 10% N2hRD-HDEL (SEQ ID NO: 4) 4.0 10%

(50) TABLE-US-00005 TABLE 2 The relative amounts of CYP1A2 obtained using the P450 reductase variants, hRD, N1hRD-M, N1hRD-HDEL and N2hRD-HDEL. Relative Amounts of Human P450 Reductase Variant CYP1A2 Obtained hRD (SEQ ID NO: 6) 1 N1hRD-M (SEQ ID NO: 9) 3.2 10% N1hRD-HDEL (SEQ ID NO: 3) 4.0 10% N2hRD-HDEL (SEQ ID NO: 3) 4.5 10%
Conclusion

(51) The relative amounts of CYP2D6 and CYP1A2 produced using the mutant human P450 reductases are appreciably higher than that obtained with the wild-type reductase, hRD.

EXAMPLE 9DIHYDROETHIDIUM ASSAY FOR ROS DETECTION

(52) The reactive oxygen species generated in yeast cells due to expression of a P450 reductase were assessed using dihydroethidium fluorescence assay. Reactive oxygen species reacts with dihydroethidium to produce ethidium bromide which binds to the nuclear DNA and emits red fluorescence. Dihydroethidine is one of the best reagents available for measuring intracellular production of reactive oxygen species. After overnight induction of a P450 reductase, the cultures were analysed for induction of reactive oxygen species. The control (wt-hRD) and test (N1hRD-M, N1hRD-HDEL, N2hRD-HDEL) yeast cultures were washed in sterile PBS and then incubated with dihydroethidium (5 M final concentration) for 30 min. After washing with sterile PBS twice (to remove the extracellular dye), the samples were transferred into 96-well black plates with transparent bottom (COSTAR). Fluorescence was measured using a BIO-TEK plate reader. Excitation and emission wavelengths were 260 and 610 nm respectively. The percent induction in the formation of reactive oxygen species was calculated by comparing with cultures where genes were not induced.

(53) The results are shown in FIG. 13 and FIG. 14. The mutant human P450 reductase, N1hRD-M gene, synthesized using yeast-biased codons produces far less ROS than the N1hRD-M gene that was isolated from the human liver cDNA library. The three mutant human P450 reductases produce far less ROS than the wild-type enzyme, hRD.

EXAMPLE 10MTT-BASED CYTOCHROME P450 REDUCTASE ASSAY

(54) The enzyme NADPH-cytochrome P450 reductase mediates the transfer of electrons from NADPH to cytochrome P450, other microsomal proteins and cytochrome c. It also catalyses the reduction of many drugs and other compounds such as potassium ferricyanide, 2,6-dichloroindopheonl, 1,1-diphenyl-2-picrylhydrazyl (DPPH), and mitomycin c. Tetrazolium salts are used extensively in cell proliferation and cytotoxicity assays, enzyme assays, histochemical procedures and bacteriological screening. In each of these processes, terazolium salts are metabolically reduced to highly coloured end products called formazans. The compound 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) is a monotetrazolium salt. The reduction of MTT is one of the most frequently used methods for measuring cell proliferation and cytotoxicity. Reduction of MTT by P450 reductase has been assessed as a method for monitoring yeast produced recombinant P450 reductase activity and the protocol was developed on the procedure published by Yim S-K, et al (Yim S-K., Y. C.-H. Ahn T., Hung H-C and Pan J-G. A continuous Spectrophotometric assay for NADPH-cytochrome P450 reductase activity using 3-(4,5 Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide. Journal of Biology and Molecular Biology 38: 366-369, 2005). The principal advantage of this substance is that the reduction of MTT can be assayed directly in the reaction medium by a continuous spectrophotometirc method. The electrons released from NADPH by P450 reductase are transferred to MTT, and then the amounts of reduced MTT is assessed spectrophotometrically by measuring the increase in A610 values that is due to the formation of blue formazan. The extinction coefficient of MTT is 11.3 mM.sup.1 cm.sup.1. This method offers the advantages of short analysis time with the use of a relatively cheap commercial substrate. The classical assay uses recombinant cytochrome c as a substrate.

(55) Solutions Used for the MTT Assay 10 mM potassium phosphate buffer: pH7.4: 8 ml of 1M K.sub.2HPO.sub.4 and 2 ml of 1M KH.sub.2PO.sub.4 add ddH.sub.2O to make up to 1 liter. 10 mM MTT: 41.4 mg of MTT (Sigma, Cat No. M2128) into 10 ml of 10 mM potassium phosphate pH7.4 to give 10 mM MTT. 100 mM potassium phosphate buffer: pH7.6: 86.6 ml of 1M K.sub.2HPO.sub.4 and 13.4 ml of 1M KH.sub.2PO.sub.4 add ddH.sub.2O to make up to 1 liter. Solution A 1 ml stock (stored at 20 C.): 131 l of 1M Magnesium Chloride solution (Sigma, Cat No.:M1028) in 1 ml ddH.sub.2O to final concentration 66 mM. NADP.sup.+ (Sigma, Cat No.: N0505, Mr 765.4) 43.5 mg, final concentration 50 mM. Hydrated salt of disodium D-Glucose-6-phosphate (Sigma, Cat No.: F7250, Mr 304.1) 172 mg, final concentration 500 mM. Solution B (stored at 20 C.): 17 U Glucose-6-phosphate dehydrogenase (Sigma, Cat No.: G6378, 250 U) in 340 l of 5 mM sodium citrate (14.7 mg/ml) (trisbasic) (Sigma, cat No.: S46410).

(56) MTT-Based P450 Reductase Assay Modified for Assessing Yeast-Derived Recombinant P450 Reductase

(57) Disposable cuvettes were used for this experiment. 850 l of potassium phosphate buffer was added to a cuvette. 100 g of yeast microsomes or 100 g of cell supernatants containing the cytosolic fraction of yeast was added to the buffer followed by 10 l of solution B. The contents were mixed gently to prevent any bubble formation in the resulting suspension. 10 l of solution A was quickly added to the cuvette, and the contents were mixed by inverting a few times. The cuvette was quickly placed into the spectrophotometer together with the blank cuvette and its contents (that contained all components as in the other test cuvette but not the microsomes or cell supernatant) and the increase in the values at 610 nm was measured for a time period of 400 seconds. The electrons released from NADPH by recombinant P450 reductase enzyme were transferred to MTT, and the ability to reduce MTT was assessed spectrophotometrically by measuring the increase in A610 values as a result of the formation of blue formazan. The rate of MTT reduction was calculated from the change in A610 values using an extinction coefficient of 11.3 mM.sup.1 cm.sup.1 and the formula, A610/min/11.3*0.1 mg/ml=mole reduced MTT/min/mg of protein.

(58) Results

(59) The results are shown in Table 3 below.

(60) TABLE-US-00006 TABLE 3 Comparative MTT reducing ability of the different reductases. M of reduced MTT/min/mg of protein hRD Variant (Relative Rates) hRD (SEQ ID NO: 6) 1 N1hRD-M (SEQ ID NO: 9) 2.5 10% N1hRD-HDEL (SEQ ID NO: 3) .sup.3 10% N2hRD-HDEL (SEQ ID NO: 4) 3.5 10%
Conclusion

(61) It seems that the new human P450 reductase variants have the potential to couple with a CYP better than the wild-type reductase.

EXAMPLE 11CYTOCHROME P450 ASSAYS FOR MEASURING SPECIFIC ACTIVITIES

(62) TABLE-US-00007 TABLE 4 Outline of the parameters used to analyse the activities of cytochrome P450 enzymes using a fluorescent plate reader (Bio-Tek Synergy HT). Conc. Final of Substrate P450 Bandwidth Conc. Dilution per of filter per of reaction Enzyme Substrate Product Excitation Emission Sensitivity reaction Substrate (l) CYP1A1 7- Resorufin 530 nm 590 nm 55 5 M DMSO 0.5 pmol Ethoxyresorufin CYP1B1 7- Resorufin 530 nm 590 nm 60 5 M DMSO 1.7 pmol Ethoxyresorufin CYP1A2 CEC CHC 400 nm 460 nm 80 16 M Acetonitrile 2 pmol CYP2D6 EOMCC CHC 400 nm 460 nm 75 10 M Acetonitrile 2.5 pmol CYP3A4 DBF Fluorescein 485 nm 528 nm 80 2 M Acetonitrile 0.5 pmol CEC = 3-Cyano-7-Ethoxycoumarin; EOMCC = Invitrogen; DBF = Dibenzylfluorescein. Protocols for enzyme assays

(63) The computer was switched on and the KC4 software (of the BioTek plate reader) was opened to select the parameters and plate layout. The plate reader machine was warmed to 37 C. 100 M of stock solutions of the compounds were used to analyse the percentage inhibition of CYPs at a final concentration of 5 M in each well.

(64) 45 l of regenerating system was prepared and pre-warmed at 37 C. (see Table 5).

(65) TABLE-US-00008 TABLE 5 The constitution of the regenerating system used per reaction in each single well for different CYPs was as follows. Enzyme Solution A Solution B Inhibitor KPi buffers Water CYP1A1 5 l 1 l 5 l 39 l 0.2M CYP1B1 5 l 1 l 5 l 39 l 0.2M CYP1A2 5 l 1 l 5 l 20 l 0.5M 19 l CYP2D6 5 l 1 l 5 l 25 l 0.2M 14 l CYP3A4 5 l 1 l 5 l 25 l 0.2M 14 l

(66) 50 l of enzyme-substrate reaction mixture was prepared and kept in an incubator at 37 C. for 10 minutes (see Table 3).

(67) TABLE-US-00009 TABLE 6 Enzyme-Substrate mixtures per reaction in each well were as follows. P450 Control Enzyme Conc. Microsome Substrate KPi buffer Water CYP1A1 0.5 l .sup.2 l 5 l 0.1 mM 42.5 l 0.1M E.R CYP1B1 0.5 l 1.7 l 5 l 0.1 mM 42.8 l 0.1M E.R CYP1A2 .sup.1 l 1.6 l 5 l 320 M 42.4 l 0.1M CEC CYP2D6 2.5 l 0.4 l 0.5 l 2 mM .sup.25 l 0.2M 21.6 l EOMCC CYP3A4 1.1 l 0.102 l 0.1 l 2 mM .sup.25 l 0.2M 23.96 l

(68) In a well of a 96-well flat-bottomed microplate, 45 l of regenerating system, 5 l of 100 potential inhibitor (from the compound library) and 50 l of enzyme/substrate mixture were added in all the wells except the wells which acted as negative controls. Instead of any compound, 5 l of 10% DMSO was added to negative control wells. After preparation of the contents of all the wells, the microplate was vortexed for a few seconds so that contents were mixed well, in each well, and incubated at 37 C. for 10 minutes. After 10 minutes, 75 l of Tris-acetonitrile (stop solution) was added to all wells using an 8-channel multi-channel pipette to stop the reaction. After that 50 l of enzyme/substrate mixture was added into a negative well. The plate was left to shake for 10 seconds and endpoint assay was run using an appropriate setting (Table 4).

(69) Reagents Used for Enzyme Activity/Inhibition Assays. 1 mM of 7-Ethoxyresorufin (ER), stored at 20 C.: MW of 7-ethoxyresorufin (ER), 241.2; 2.412 mg of ER in 100% DMSO. This solution was further diluted to 0.1 mM in 1% DMSO on the day of use. 10 mM 3-Cyano-7-ethoxycoumarin (CEC), stored at 20 C.: MW of CEC, 215.2; 2.152 mg of CEC in 100% acetonitrile. This was further diluted to 0.32 M in 1% DMSO on the day of use. 2 mM 7-ethylmethyloxy-3-cyanocoumarin (EOMCC), stored at 20 C.: MW of EOMCC, 245.2; 0.1 mg of EOMCC in 100% acetonitrile. 2 mM Dibenzylfluorescein (DBF), stored at 20 C.: MW of DBF, 512.55; 2.06 mg of DBF in 100% acetonitrile. 100 mM (0.1 M) potassium phosphate buffer (KPi) at pH 7.4: 0.3 ml of 1.0 M K.sub.2HPO.sub.4+4.7 ml of 1.0 M KH.sub.2PO.sub.4 were mixed and made up to 50 ml with distilled water. 100 mM (0.2 M) potassium phosphate buffer (KPi) at pH 7.4: 0.6 ml of 1.0M K.sub.2HPO.sub.4+9.4 ml of 1.0M KH.sub.2PO.sub.4 were mixed and made up to 50 ml with distilled water. 500 mM (0.5 M) potassium phosphate buffer (KPi) at pH 7.4: 1.5 ml of 1.0 M K.sub.2HPO.sub.4+23.5 ml of 1.0 M KH.sub.2PO.sub.4 were mixed and made up to 50 ml with distilled water. Solution A stored at 20 C.: 183 mg of NADP.sup.++183 mg of glucose-6-phosphate+654 l of 1.0 M Magnesium chloride solution were mixed in a sterile tube containing 9.15 ml of distilled water and the mixture was aliquoted into 1.5 ml eppendorf tubes for storage at 20 C. Solution B, stored at 20 C.: 250 Units of glucose-6-phosphate dehydrogenase+6.25 ml of 5 mM sodium citrate, mixed in a tube and made up to 10 ml with distilled water. 10% DMSO: 1 ml of 100% DMSO was diluted in 9 ml of distilled water and stored in a dark place at room temperature. 1% DMSO: 1 ml of 10% DMSO was diluted in 9 ml of distilled water and stored in a dark place. Tris-acetonitrile (Stop solution): 100 ml of 0.5 M Tris-HCl+400 ml of 80% acetonitrile.
Results

(70) TABLE-US-00010 TABLE 7 Comparative level of CYP2D6 enzyme produced using the different reductases. pmoles of CYP2D6/mg of total protein hRD Variant (Relative Amounts) hRD (SEQ ID NO: 6) 1 N1hRD-M (SEQ ID NO: 9) 4 10% N1hRD-HDEL (SEQ ID NO: 3) 6 10% N2hRD-HDEL (SEQ ID NO: 4) 6 10%

(71) TABLE-US-00011 TABLE 7 Comparative level of CYP1A2 enzyme produced using the different reductases. pmoles of CYP1A2/mg of total protein hRD Variant (Relative Amounts) hRD (SEQ ID NO: 6) 1 N1hRD-M (SEQ ID NO: 9) 4 10% N1hRD-HDEL (SEQ ID NO: 3) 6 10% N2hRD-HDEL (SEQ ID NO: 4) 6 10%
Conclusion

(72) It appears that the variant human P450 reductases have a far better ability to activate CYP2D6 and CYP1A2 than the wild-type enzyme.

(73) While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.