High-level expression of human cytochrome P450 1A2 by co-expression with human molecular chaperone HDJ-1(Hsp40)

Protein Expression and PuriWcation 36 (2004) 48–52 www.elsevier.com/locate/yprep

High-level expression of human cytochrome P450 1A2 by co-expression with human molecular chaperone HDJ-1(Hsp40) Taeho Ahn,a,¤ Siyoung Yang,b and Chul-Ho Yunc,¤ a

Department of Biochemistry, College of Veterinary Medicine, Chonnam National University, Gwangju 500-757, Republic of Korea b Department of Molecular Medicine, Chonnam National University, Gwangju 500-757, Republic of Korea c School of Biological Sciences and Technology, Chonnam National University, Gwangju 500-757, Republic of Korea Received 11 December 2003, and in revised form 9 March 2004 Available online 14 May 2004

Abstract Human cytochrome P450 (CYP) 1A2 is of great interest because of its important roles in the oxidation of numerous drugs and carcinogens. HDJ-1, a molecular chaperone in human, is known to assist the correct folding of unfolded proteins. To achieve high yield of recombinant human CYP1A2 in Escherichia coli, the CYP1A2 encoding gene was co-expressed with the chaperone HDJ-1 under the control of an inducible tac promoter in bicistronic format. Expression level of CYP1A2 in the bicistronic construct reached up to 520 nmol/liter culture within 16 h at 37 °C, which is 3.4-fold increase compared to the expression yield of CYP1A2 alone without HDJ-1. By co-expression with HDJ-1, the catalytic activity of CYP1A2 was also increased 5.5-fold. The activity increase seems to be associated with the increase of CYP production at whole cell level. The present over-expression system may be useful for rapid production of large amounts of active human CYP1A2 in E. coli.  2004 Elsevier Inc. All rights reserved. Keywords: Molecular chaperone HDJ-1; Human CYP1A2; CYP1A2 expression

Cytochromes P450 (CYP or P450)1 are the major enzymes involved in the oxidation of xenobiotic chemicals and endogenous substrates [1] and multiple forms of CYPs are present in mammals [2]. Human CYP1A2, located predominantly in the liver, participates in the metabolism of a variety of compounds including the activation of potentially carcinogenic aryl and heterocyclic amines [3]. It is also of considerable interest due to the diversity of reactions it metabolizes [4] and its relevance to cancer risk [5]. Recombinant human CYP enzymes have been proved to be useful for drug metabolism research and thereby ¤ Corresponding authors. Fax: +82-62-530-2809 (T. Ahn), +82-62530-2194 (C.-H. Yun). E-mail addresses: [email protected] (T. Ahn), chyun@ chonnam.ac.kr (C.-H. Yun). 1 Abbreviations used: CYP or P450, cytochrome P450; E. coli, Escherichia coli; PCR, polymerase chain reaction; IPTG, isopropyl-
D-thiogalactopyranoside; -ALA, -aminolevulinic acid; Chaps, 3-[(3cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate.

1046-5928/$ - see front matter  2004 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2004.03.005

many heterologous expression systems have been developed [6]. Bacterial expression systems, especially Escherichia coli cells, have also been widely used for the production of various CYP enzymes in order to obtain high yield of the proteins. Moreover, to achieve maximal catalytic activity as well as protein expression, various strategies are employed such as co-expression of CYP with NADPH-P450 reductase [7], N-terminal modiWcation [8], and use of fusion proteins [9]. Similarly, it was reported that the expression of CYP3A7 was elevated by the presence of molecular chaperone GroEL, known to assist the correct folding of proteins in E. coli [10]. In this study, we provide an over-expression system for human CYP1A2 in E. coli achieving high production of the CYP1A2 enzyme by co-expression with HDJ-1, a molecular chaperone in human. HDJ-1 (also called Hsp40) is a human homologue of bacterial heat shock protein DnaJ [11] and is also known to regulate the formation of protein aggregation, protein folding, and translocation [12,13].

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Materials and methods Materials NADP+, glucose-6-phosphate, glucose-6-phosphate dehydrogenase, and 7-ethoxyresoruWn were obtained from Sigma Chemical (St. Louis, MO). Recombinant rat NADPH-P450 reductase was expressed in E. coli and puriWed as described [14]. Construction of CYP expression plasmids The cDNA for human CYP1A2 with N-terminal truncation [15], which was kindly provided by Professor

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F. Peter Guengerich (Vanderbilt University, Nashville, TN, USA), was subcloned into the expression vector pCW to generate pCYP1A2. HDJ-1 gene in the pET21d HDJ-1, an expression vector for HDJ-1 in E. coli, was ampliWed by PCR. To construct co-expression vector for CYP1A2 and HDJ-1, the PCR ampliWcation was designed to include HindIII restriction enzyme site followed by ribosomal binding site and six nucleotide spacer (forward: 5⬘ AAGCTT GAAGGAGGGCTAA CATGGGTAAAGACTAC 3⬘; reverse: 5⬘ AAGCTTC TATATTGGAAGAACCTG 3⬘) as shown in Fig. 1. PCR was performed with Pfu polymerase (Stratagene, UK). The mixture was preincubated for 5 min at 94 °C before the addition of the polymerase, followed by

Fig. 1. Construction of co-expression plasmid for human CYP1A2 and HDJ-1 in bicistronic format.

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ampliWcation for 30 cycles: 94 °C for 90 s, 60 °C for 90 s, and 72 °C for 3 min. The resulting PCR product was puriWed, digested with HindIII, and then ligated into pCW1A2 vector treated with HindIII. The nucleotide sequence of the entire region including CYP and HDJ-1 was analyzed by dideoxy sequencing [16]. Expression of recombinant CYP1A2 and preparation of membranes Escherichia coli DH5F⬘IQ cells were transformed with the expression plasmid. Transformed cells were grown in TerriWc Broth (TB) containing 0.2% bactopeptone (w/v). TB was supplemented with 100 g ampicillin/ml, 1.0 mM thiamine, trace elements [17], 50 M FeCl3, 1 mM MgCl2, and 2.5 mM (NH4)2SO4. When the optical density of the culture at 600 nm was approximately 0.4–0.5, protein expression was induced by adding 1 mM IPTG and 1.5 mM -ALA. At every 3 h, a portion of culture was sampled and expression level of CYP was quantitated by Fe2+–CO versus Fe2+ diVerence spectra [18]. Expression was allowed to proceed for 24 h at 37 °C. Bacterial membrane fractions were prepared as described previously [15].

Results and discussion Several strategies for the expression of recombinant human CYPs in bacteria have been developed and widely used [6]. It was shown that GroEL, a molecular chaperone, increases the expression level of CYP3A7 in E. coli [10]. As a result of searching for other chaperones that have eVects on the level of CYP production, a bicistronic construct expressing both CYP1A2 and HDJ-1 was prepared as outlined in Fig. 1. Fig. 2 shows the induction time-dependent expression level of recombinant CYP1A2 in E. coli cells at 37 °C. Regardless of the presence or absence of chaperone HDJ-1, the CYP production was elevated with increasing induction time and maximal yield was obtained at around 12–16 h when assayed by Fe2+–CO versus Fe2+ diVerence spectra of whole E. coli cells. However, further incubation decreases the level of CYP production, but the reason is not clear.

Enzymatic assay Whole cells and membrane fractions were analyzed for expressed CYP concentrations by Fe2+–CO versus Fe2+ diVerence spectroscopy as described [18], in 100 mM Tris–Cl, pH 7.4, containing 20% glycerol (v/v), 10 mM Chaps, and 1 mM Na-EDTA, using a Shimadzu UV-1650 PC spectrophotometer. The catalytic activity of CYP1A2 was measured with membrane fractions. EthoxyresoruWn O-deethylation activity was determined for CYP1A2, as described elsewhere [19]. The activity assay was performed in 100 mM potassium phosphate buVer (pH 7.4). The reaction volume was 500 l. Membrane fraction (0.4 M CYP1A2) and NADPH-cytochrome P450 reductase (2 M) were mixed in the presence of 7-ethoxyresoruWn as a substrate. The reaction was started by adding an NADPH-generating system, and after incubating the sample at 30 °C for 10 min, the reaction was stopped by adding 500 l of cold methanol. The formation of resoruWn was monitored spectroXuorometrically [19]. Other methods Protein concentrations were estimated using a bicinchoninic acid procedure according to the manufacturer's directions (Pierce, Rockford, IL). Rabbit antihuman CYP1A2 was used in the immunoblotting experiments presented here. The speciWcity and properties of similar preparations have been reported elsewhere [20]. The antiserum was adsorbed with E. coli proteins before use to remove background staining.

Fig. 2. Induction time-dependent expression level of recombinant human CYP1A2. (A) The Fe2+–CO versus Fe2+ diVerence spectra with increasing induction time showing increased expression of CYP1A2 from the construct of CYP1A2-HDJ-1 co-expression in whole E. coli cells. Each spectrum was obtained after incubation at 3, 6, 9, and 12 h. In (B), error bars represent §SD with three independent determinations.

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Table 1 Expression yield of recombinant human CYP1A2 with and without HDJ-1 expressed in E. colia Construct

CYP1A2 alone CYP1A2 + HDJ-1 a b

Expression level Whole cells (nmol/liter culture)

Membranes (nmol/liter culture)

152 § 22b 522 § 49

59 § 4 250 § 26

Membrane CYP content (nmol/mg protein) 0.16 § 0.04 0.65 § 0.07

Expression was carried out at 37 °C for 16 h in E. coli DH5F⬘IQ in the presence of 1.0 mM IPTG and 1.5 mM -ALA. Results are expressed as means § SD with three independent experiments.

Fig. 3. Immunoblot analysis of CYP1A2 expression in whole E. coli cells as induction over time. The blot was probed with rabbit anti-human CYP1A2 primary antibody and HRP-linked rat anti-rabbit IgG secondary antibody.

By co-expression with HDJ-1, the production level of CYP1A2 in whole cells increased »3.4-fold compared to that of expressed CYP1A2 without HDJ-1. Concomitantly, the incorporation of CYP1A2 into membranes was also stimulated, which reminds us the result that minor modiWcations of the N-terminal part of CYP1A2 allow the elevated expression of membrane bound CYP1A2 [15]. Table 1 summarizes the maximal production level of CYP1A2. More interestingly, when HDJ-1 was co-expressed, the induction time-dependent decrease of production level was less signiWcant compared to the case of CYP1A2 alone. At present, however, it is not clear how HDJ-1 exerts its function to make CYP1A2 resistant against the induction time-dependent decrease of CYP expression. The expression of CYP1A2 was also conWrmed by Western blot analysis (Fig. 3). These results suggest the possibility that the expression of holoenzyme of CYP1A2 is dependent on the status of protein folding and HDJ-1 assists the correct folding of the CYP1A2 in E. coli. As a control experiment, the production of HDJ-1 was also checked using antiserum against the protein (results not shown). We assayed the catalytic activity of CYP1A2 with membrane fractions and the results are summarized in Table 2. As expected, the activity was stimulated by »5.5-fold by co-expression of HDJ-1 compared with the value for CYP alone. The activity increase seems to be associated with the increase of CYP production at the whole cell level. Based on the values in Tables 1 and 2, the activity of CYP1A2 expressed without chaperone is 1.5 nmol/min/nmol of CYP and that of the CYP1A2 coexpressed form is 2 nmol/min/nmol of CYP. This is likely to be related to a better incorporation of heme in the CYP. This result suggests that HDJ-1 is involved in the

Table 2 Activities of recombinant human CYP1A2 with and without HDJ-1 expressed in E. coli Construct

7-EthoxyresoruWn O-deethylation activitya (nmol product formed/min/mg protein)

CYP1A2 alone CYP1A2 + HDJ-1

0.24 § 0.08b 1.31 § 0.19

a Activity was determined with bacterial membranes and represented in nmol/min/mg protein. b Results are expressed as means § SD with three independent experiments.

correct folding of CYP1A2 as well as its incorporation into membranes as suggested.

Conclusion We report here the successful construction of expression system for the production of recombinant human CYP1A2 in E. coli. Expression level and catalytic activity were enhanced by co-expression with human molecular chaperone HDJ-1 in bicistronic format. The present system is very rapid when compared with other bacterial expression methods reported, which usually employ long culture time (48–72 h) at lower temperature (usually under 30 °C). The production level of CYP1A2 and the catalytic activity were also compatible.

Acknowledgments This work was supported by Grant No. R5-2003-00010504-0 from Korea Science and Engineering Foundation

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to T.A. We thank Professor J.H. Song (Sungkyunkwan University, Suwon, Korea) for providing the expression vector for HDJ-1 (pET21d HDJ-1).

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