Proc. Natl. Acad. Sci. USA Vol. 89, pp. 10817-10821, November 1992 Biochemistry High-level expression in Escherichia coli of enzymatically active fusion proteins containing the domains of mammalian cytochromes P450 and NADPH-P450 reductase flavoprotein (hybrid protein/steroid 17a-hydroxylation/to-oxidation) CHARLES W. FISHER, MANJUNATH S. SHET, DEBORAH L. CAUDLE, CHERYL A. MARTIN-WIXTROM, AND RONALD W. ESTABROOK Department of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235 Contributed by Ronald W. Estabrook, August 13, 1992 ABSTRACT This report describes the properties of two 102 is the most enzymatically active form ofany known P450 mammalian cytochromes P450 that have been expressed at high (turnover number > 1500/min). levels inEschenichia coli as enzymatically active fusion proteins Murakami et al. (4) genetically engineered the cDNA ofrat containing the flavoprotein domain ofrat NADPH-cytochrome liver P450c (P450 lA1) with the cDNA of rat NADPH-P450 P450 reductase (EC 1.6.2.4). Fusion proteins were prepared by reductase to construct a P450 fusion protein which they engineering the cDNAs for the steroid-metabolizing bovine expressed in yeast. Subsequent studies by this group (5) adrenal P450 17A with the cDNA for rat liver NADPH-P450 extended their technique to the expression in yeast of fusion reductase with the introduction of a Ser-Thr linker to give a proteins of P450s active in steroid metabolism by using the protein we have named rF450[mBovl7A/mRatOR]Ll. Simi- cDNAs ofbovine P450 17A or bovine P450 21 and the cDNA larly, the cDNA for the w-hydroxylase of rat liver (P450 4A1) of the yeast flavoprotein NADPH-P450 reductase. An en- was linked with the cDNA for rat liver NADPH-P450 reductase hanced enzymatic activity for the fusion protein containing to give rF450[mRat4Al/mRatORjLl. A procedure involving bovine P450 17A in the hydroxylation of progesterone was disruption of transformed E. coli by sonication, isolation of reported (6). membranes by differential centrifugation, solubilization with Studies of P450s have been greatly facilitated by the detergent, and affinity chromatography provided sicant successful application ofheterologous expression techniques amounts of purified fusion proteins of 118 kDa. The purified (7). This approach has been particularly useful when applied fusion proteins had turnover numbers for the metabolism of to P450s engineered for site-specific mutations (8) or for the steroids (rF450[mBovl7A/mRatORJLl) or fatty acids generation of large quantities of difficult-to-obtain biologi- (rF450[mRat4Al/mRatOR]Ll) ranging from 10/min to cally active proteins, such as human P450s (9). 30/min in the absence of added phospholipid. Addition of Recently, the high-level expression in Escherichia coli of purified rat liver cytochrome b5 stimulated the 17,20-lyase bovine adrenal P450 17A (10) and human liver P450 1A2 (9) reaction for the conversion of 17-hydroxypregnenolone to has been described. These studies are extended here to the dehydroepiandrosterone, and addition of purified rat expression, purification, and enzymatic characterization of NADPH-cytochrome P450 reductase enhanced the formation two fusion proteins containing the functional domains of of t - 1 metabolites from lauric and arachidonic acids. different microsomal P450s linked to the microsomal flavo- NADPH oxidation was tightly coupled to substrate hydroxy- protein domain of NADPH-P450 reductase. We introduce a lation with the purified fusion proteins. nomenclature for these fusion proteins, rF450[mBovl7A/ mRatORJL1 and rF450[mRat4Al/mRatOR]Ll, where r = Cytochromes P450 (P450s) of mammalian tissues can be recombinant; F = fused; 450 = P450; m = modified cDNA; categorized into two groups (1) depending on their intracel- Bov or Rat = species; 17A or4A1 = family name ofP450; OR lular compartmentation: P450s associated with the endoplas- = NADPH-P450 reductase; and Li = linker type. mic reticulum are one type, and these microsomal P450s require only an NADPH-reactive, FAD- and FMN- MATERIALS AND METHODS containing flavoprotein for the transfer of electrons from Plasmids and E. coli Strains Used. A construct expressing NADPH to a P450; P450s associated with mitochondria are bovine P450 17A in vector pCWori+ (pCWmodl7) was ob- a second type, and these mitochondrial P450s function with tained from the laboratory of Michael Waterman (10); a a mini electron-transfer chain composed of an NADPH- 2.1-kilobase (kb) insert in pUC19 containing the cDNA for reactive FAD-containing flavoprotein and a P450-reactive the open reading frame for rat liver P450 4A1 was obtained iron-sulfur protein. Many different P450s have been used to from G. Gordon Gibson (11), University ofSurrey, U.K.; the reconstitute enzymatic activities by incubating a purified cDNA for rat liver NADPH-P450 reductase (pOR263) was P450 with its designated purified electron-transfer partner(s) obtained from Charles Kasper (12), University ofWisconsin. in the presence of a suitable phospholipid (2). Recombinant DNA Manipulations. Initial experiments were Miura and Fulco (3) were the first to describe the presence directed to the construction by PCR mutagenesis ofa plasmid of a P450 as a fusion protein-i.e., a single protein containing encoding bovine P450 17A fused to rat liver NADPH-P450 both the heme domain of a P450 and the flavoprotein domain reductase. The coding sequence of the amino terminus of equivalent to the microsomal FAD- and FMN-containing bovine P450 17A had been modified (10). Mutagenesis was NADPH-P450 reductase. They isolated and purified this performed to modify the coding sequence for the carboxyl soluble P450BM-3, named P450 102, from Bacillus megaterium terminus of bovine P450 17A and the coding sequence for the and characterized it as an c-hydroxylase of fatty acids. P450 amino terminus of rat liver NADPH-P450 reductase in a manner similar to that described by Murakami et al. (4) in The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]- in accordance with 18 U.S.C. §1734 solely to indicate this fact. 1-propanesulfonate; 2',5'-ADP, adenosine 2',5'-bisphosphate. 10817 10818 Biochemistry: Fisher et al. Proc. Natl. Acad. Sci. USA 89 (1992) order to allow fusion of these sequences with one encoding a 509 57 dipeptide linker, Ser-Thr (Fig. 1). To digest the plasmid A Rat P450 4A1 Rat NADPH-P460 OR LysLysLeuHisSerThrIleGlnThr (pCWmodl7) at the methylated Stu I site the plasmid was GGTAGCACCCC=CGACTATCCAAACA transformed into the dcm- E. coli strain GM48 and plasmid Sal I°IXho I" A (bp) fragment was deleted DNA was prepared. 40-base-pair Bovine P450 17A Rat P4W 4A1 by digesting the plasmid with Stu I and HindIII. Rat liver B NADPH-P450 reductase cDNA (pOR263) was amplified by 23 24 25 26 27 Met Ala Leu Leu Leu Ala Val Phe Leu Gly Lou Leu Lou Lou PCR using primers deleting the amino-terminal membrane- ATG GCT CTG TTA TTA GCA GTT TTT CTG GTT CTG CTG CTG GTC anchoring region and incorporating a Sal I site encoding Ser-Thr as a linker. The 3' reductase primer incorporated a FIG. 2. Modifications of the amino and carboxyl termini of rat HindIII site after the TAG stop codon. The PCR fragment was P450 4A1. (A) Region offusion between the rat P450 4A1 domain and the rat NADPH-cytochrome P450 domain (cf. Fig. 1). The hybrid Sal subcloned into the Sma I site of pTZ19R. The 5' and 3' ends I/Xho I site is underlined. (B) Modification of the amino terminus of of the amplified sequence were sequenced to the two internal P450 4A1 and fusion to the 27-bp fragment encoding 9 amino acids Nco I sites. Bovine P450 17A was amplified by PCR with a 3' of modified bovine P450 17A. Numbers in bold are those of rat P450 primer deleting the TGA stop codon and incorporating the 4A1. Alignment ofthe amino-terminal regions ofthe modified bovine same Sal I site encoding Ser-Thr as the reductase 5' primer. 17A (10) and rat P450 4A1 was based upon the data of Nelson and The PCR product was digested with BamHI and Sal L. The Sal Strobel (13). I-HindIII reductase domain was ligated to the BamHI-Sal I P450 4A1. The properties of these fusion proteins will be P450 domain fragment and pTZ19R digested with BamHI and described in subsequent publications. HindIII. A clone containing the BamHI-HindIII fragment was Cell Growth and Harvesting. Growth conditions for the sequenced from the Stu I site to the Sal I site. The positive expression of P450 fusion proteins were similar to those clone was digested with Nco I to remove the 1.4-kb internal described (9, 10). The cells were harvested, washed with 10 Nco I-Nco I fragment from the PCR-generated reductase mM potassium phosphate buffer, pH 7.5/0.15 M NaCl, domain. The 1.4-kb Nco I fragment was isolated from the suspended in a Dounce homogenizer with 2 volumes (vol/wt) plasmid preparation of the original rat liver reductase cDNA. ofTSE buffer (75 mM Tris HCI, pH 7.5/250mM sucrose/0.25 The Nco I fragment was ligated with the Nco I-deleted vector mM EDTA), and divided into 100-ml aliquots (33 g wet construct. A construct with a replaced Nco I fragment in the weight) and frozen at -80°C. correct orientation was identified and transformed into E. coli Cell Disruption and Preparation of Membranes. Mem- GM48, and the resulting plasmid preparation was digested branes were prepared from 100 ml of thawed cells to which with Stu I and HindIl. The 1.9-kb Stu 1-HindIII fragment was 0.5 ml of200 mM phenylmethanesulfonyl fluoride was added. ligated with the original pCWmodl7 digested with Stu I and Cells were disrupted at 0°C by sonic treatment with a Fisher HindIlI.