Baculovirus expression and characterization of catalytically active horseradish peroxidase

ARCHIVES

OF BIOCHEMISTRY

AND

BIOPHYSICS

Vol. 297, No. 1, August 15, pp. 61-72, 1992

Baculovirus Expression and Characterization Catalytically Active Horseradish Peroxidasel Christa Department

Hartmann

and Paul R. Ortiz

of Pharmaceutical

Received February

Chemistry,

of

de Montellano2

School of Pharmacy,

University

of California,

San Francisco, California

94143-0446

27, 1992, and in revised form April 9, 1992

Studies of horseradish peroxidase (HRP), a prototypical enzyme, have provided much of the information that is available on the mechanisms and functions of hemoprotein peroxidases. HRP itself is widely used in biotechnological applications. Further progress in defining the structure and function of the enzyme, however, requires its expression in a heterologous system. We report here baculovirus-mediated, high yield expression of a synthetic gene for HRP in Spodoptera frugiperda cell culture. Expression of the soluble, glycosylated protein requires the 5’-leader sequence of the native gene. Recombinant horseradish peroxidase reacts with H202 to give compound I, II, and III spectra and a guaiacol oxidation activity, identical to those of the native enzyme. The integrity of the recombinant active site is confirmed by NMR spectroscopy and by catalytic reaction with ethylhydrazine to give a stabilized isoporphyrin that decays exclusively to b-meso-ethylheme. Furthermore, thioanisoles are oxidized by recombinant and native HRP with the same enantiomeric specificity. HRP expressed in a baculovirus system, despite probable differences in glycosylation, is essentially identical to the native enzyme. 0 1992 Academic Press, Inc.

Hemoprotein peroxidases are a widely distributed class of enzymes that catalyze the oxidation of diverse substrates at the expense of hydrogen peroxide. Horseradish peroxidase (HRP),3 from the root of Armoracia rusticanu, and cytochrome c peroxidase (CcP), from Saccharomyces cereuisiae, are the best characterized of these enzymes (l3). The mammalian enzymes, including myeloperoxidase ’ This work was supported by Grant GM32488 and Fellowship Award GM13578 (to C.H.) from the National Institutes of Health. ’ To whom correspondence should be addressed. 3 Abbreviations used: HRP, horseradish peroxidase; CcP, cytochrome c peroxidase; FPLC, fast protein liquid chromatography; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; FCS, fetal calf serum. 0003.9861/92 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

(4)) thyroid peroxidase (5)) eosinophil peroxidase (6)) lactoperoxidase (7), andprostaglandin synthase (8), are sufficiently less tractable than HRP and CcP so that most of what is known about the structure and mechanism of peroxidases is based on work with the plant and fungal enzymes. HRP has been characterized by many chemical and spectroscopic methods, including NMR (g-11), but not yet by X-ray crystallography. A crystal structure is available for CcP (12) but the mechanistic differences between HRP and CcP, which parallel differences observed in the mammalian peroxidases, make continued efforts to characterize both prototypical peroxidases essential. HRP appears to have the same catalytic histidine and arginine residues as CcP but differs critically in that reaction of HRP with HzOz gives a catalytic (“compound I”) species in which a ferry1 (Fe(IV) = 0) complex is couradical cation (13-17), whereas the pled with a porphyrin ferry1 complex of CcP is associated with a protein radical (17-20). One-electron transfer quenches the porphyrin or protein radical, yielding analogous compound II species for both enzymes which only retain the ferry1 complex (l-4, 17). The observation that HRP mimics some reactions of lactoperoxidase and thyroid peroxidase, whereas CcP mimics others, is presumably related to the fact that the two possible compound I structures appear to coexist in these mammalian enzymes (21). The amino acid sequence of HRP isozyme c has been determined by both amino acid sequencing and cDNA methods (22,23). The protein is stabilized by four disulfide bonds and is highly glycosylated, approximately 21% of its molecular weight (-43,000) being accounted for by carbohydrate residues attached to eight glycosylation sites (22). These two properties complicate its recombinant expression in bacteria because disulfide bonds are difficult to form in the reducing bacterial environment and bacteria do not glycosylate proteins. In preliminary work, we found that HRP could be expressed in large amounts as inclusion bodies from which it was difficult to recover significant yields of the catalytically active enzyme (unpublished). During the course of our studies, three laboratories re61

62

HARTMANN

(1)

Hpal

(2)

BamHl

(3)

Ligate

AND

ORTIZ

Digestion Digestion with

ollgos

and pVL1392

DE MONTELLANO

(1)

Pstl

(2)

BamHl

Digestion

(3)

Ligate

Digestion with

oligos

and HRP gene

Hpalsite FIG. 1. Construction of transfer plasmid pVLHRP2. The transfer vector pVL1392 contains a unique PstI site at the 5’ end of the AcMNPV polyhedron gene and a unique BumHI in the polylinker multiple cloning site. The plasmid pUC-HRP contains the synthetic HRP gene. To prepare the transfer vector pVLHRP2, the HRP gene was excised from pUC-HRP by digestion with HpaI and BarnHI, followed by ligation with annealed synthetic oligonucleotides and the pVL1392 plasmid digested with PstI and BanHI. The resulting plasmid, pVLHRP2, encodes the entire HRP coding region, including the N-terminal leader sequence.

ported the expression of HRP in Escherichia coli (24-26), but in only one case could the expressed protein be obtained in active form (26). The active protein was obtained in low yield (- 100 pg/liter) after refolding in the presence of 2 M urea and 5 mM Ca2+ (26). We have been unable to reproduce these folding experiments. We report here expression and overproduction of HRP isozyme c in insect tissue culture using a baculovirus transfer vector, a system that has been quite successful for the high level expression of other difficult eukaryotic proteins (27). The expressed enzyme has been purified and has been characterized by a variety of spectroscopic, catalytic, and chemical techniques that establish its structural integrity. EXPERIMENTAL

PROCEDURES

Materials. Thioanisole, para-methoxythioanisole, phenylmethylsulfonyl fluoride, pepstatin, leupeptin, and aprotinin were purchased from Aldrich (Milwaukee, WI). Horseradish peroxidase (Type VI),

guaiacol, and ammonium sulfate were from Sigma (St. Louis, MO), amannosidase was from Boehringer-Mannheim (Indianapolis, IN), and N-glycosylase F was from Genzyme (Boston, MA). Restriction enzymes were purchased from New England Biolabs (Beverley, MA), BoehringerMannheim, or Bethesda Research Laboratories (Gaithersburg, MD). Oligonucleotides were synthesized at the University of California, San Francisco, Biomolecular Resource Center using an Applied Biosystems 380B DNA synthesizer. Insect tissue culture media was from JRH Biosciences (Lenexa, KA). Heat-inactivated fetal calf serum, penicillin, streptomycin, and E. coli strain DH5a were obtained from the University of California, San Francisco, Cell Culture Facility. DNA mini- and midiprep columns were obtained from Qiagen (Chatsworth, CA). Buffers were made with deionized, glass-distilled water that had been stirred at least 3 h with 1 g/liter Chelex 100 beads (Bio-Rad, Richmond, CA). General methods. Plasmid purifications were carried out using a modification of the procedure of Maniatis et al. (28) or by passage through Qiagen mini- or midi-prep columns. Oligonucleotides were purified on PAGE 12% acrylamide/8 M urea gels (28). DNA was eluted from the gels in 0.5 M ammonium acetate and desalted by passage through a Pharmacia (Piscataway, NJ) NAP-25 column with water as eluant. HRP assay incubations were carried out in 50 mM sodium phosphate buffer, pH 7.2, with 5 mM guaiacol and 0.6 mM HZOZ.

BACULOVIRUS

A280

0

EXPRESSION

OF HORSERADISH

63

PEROXIDASE

0.05

6

1

12

18

1 24

I

1 30

1 36

FRACTION FIG. 2. Purification of active recombinant HRP from Spodoptera frugiperda expression medium. Following ammonium sulfate precipitation and size exclusion chromatography, the enzyme sample was dialyzed against 20 mM sodium acetate buffer, pH 4.3, and absorbed onto a Mono S HR lO/lO FPLC cation exchange column, equilibrated with the same buffer. Elution was with a linear gradient, O-l M NaCl, at a flow rate of 2 ml/fraction/min. The activity and purification factors for each step in the purification are shown in Table I. Ados(-); A&-- -).

Analytical techniques. Thioanisole and para-methoxythioanisole oxidations by recombinant and native HRP were carried out at room temperature for 1 hand typically included 10 pM enzyme, 5 mM substrate, and 1 mM H,Ox in 0.5 ml 50 mM sodium phosphate buffer, pH 7.2. The peroxide was added in aliquots every 5 min over a 30.min period. The

TABLE Purification

of Recombinant

frugiperda

Purification

step

Medium 40-90s ammonium sulfate Mono S (FPLC) Mono Q (FPLC)

(Sf9)

I HRP from Cells”

Peroxidase activityb (~mol/min/nmol) 0.211 1.06 4.21 19.6

Spodoptera

( A~mRjAm )

Purification factor (-fold)

0.294

0.267 1.61 3.01

5 20 100

’ The purification was done starting from 600 ml of expression medium. b One unit will produce 1 pmol of tetraguaiacol/min in the presence of 0.6 mM H,O,, 5 mM guaiacol, 50 mM sodium phosphate buffer, pH 7.2, at

25°C.

enzyme was allowed to react with substrates for an additional 15 min and was then extracted into 200 ~1 CHzClz. The organic layer was evaporated and the products were resuspended in 160 ~1 hexane. Products were analyzed by isocratic HPLC on a Hewlett-Packard Model 1090A system using a Daicel OB chiral column eluted with 80:20 hexane:isopropanol. &meso-Ethylheme extracted from ethylhydrazine-modified recombinant and native HRP was analyzed as previously described (29, 30). Guaiacol assays were performed on a Hewlett-Packard Model 8450A diode array spectrophotometer. uv-visible absorption spectra were acquired on a Hewlett-Packard Model 8450A or an Aminco DW-2000 spectrophotometer. NMR characterization of native and recombinant HRP. Solutions for proton NMR studies were 0.17 mM for recombinant HRP in 50 mM Na,HPO,, pH 7.2, and 3.0 mM for the native enzyme in *HsO, pH 7.0. The solution pH, adjusted using 5.5 M *HCl or 0.2 M NaO’H, was measured with a Fisher Scientific Accumet 925 pH meter. The pH was not corrected for the isotope effect. HRPCN was formed by the addition of at least 2 eq of KCN to the HRP solution. High field NMR spectra were acquired on a General Electric NMR B-500 spectrometer. NMR spectra of the resting state enzyme were acquired on a General Electric B-300 spectrometer. Spectra consisted of 20,000 pulses using 8K over a 30,000Hz bandwidth (high field spectra) and 2K over a 60,000-Hz bandwidth (resting state) using a 6-~s 90” pulse. The residual water peak in the high field spectra was suppressed by a 200-ms presaturation pulse; signal to noise was improved by introducing lo- to 50.Hz line broadening. Peak shifts were referenced to the residual water line. Chemical shifts are reported in parts per million.

64

HARTMANN

1

2

3

45

6

AND

ORTIZ

DE MONTELLANO

-106 * 80

-106 480

-

49.5

-

49.5

*

32.5

-

32.5

-

27.5

-

21.5

-

18.5

-

18.5

7

FIG. 3. SDS-PAGE of recombinant HRP. Lanes: 1 and 7, molecular weight standards; 2, FPLC-purified recombinant HRP from cell lysate; 3, FPLC-purified recombinant HRP isolated from the medium; 4, Sf9 cell extract from 5-day expression with pVLHRP2; 5, noninfected midlog SKI cells: 6, HRP (Sigma, Type VI). Approximately 20-40 pg of protein was loaded into each lane.

Tissue culture. Spodoptera frugiperda St9 cells were a generous gift from Dr. Harold Varmus in the Department of Microbiology, University of California, San Francisco. Cells were maintained in monolayers or spinner flasks at 27°C in TNM-FH medium containing 10% heat-inactivated FCS. Cells were routinely subcultured every 2-3 days. Vectors. Plasmid pUC-HRP, containing the synthetic gene cloned into pUC19, was purchased from British Biotechnologies, Ltd. The synthetic gene optimizes mammalian codon usage and contains a large number of evenly distributed, unique restriction sites. The sequence of the commercial gene is based on the amino acid sequence of Welinder (22), recently confirmed by the cDNA sequence from horseradish root (23). The synthetic gene does not include the endogenous 5’-leader sequence found in the plant gene. Plasmid pVL1392 was kindly provided by Dr. Harold Varmus after a licensing agreement was signed with Dr. Max Summers (Texas A & M University). The plasmid pVLHRP1 was constructed by inserting the HRP gene between the PstI and BamHI sites of the polylinker multiple cloning site [5’-P&I-BgnII-XrnuII-NotIEcoRI-XbaI-SmaI-BarnHI-3’1. The PstI site was blunt-ended with T4 polymerase followed by digestion with BarnHI. The vector was gel purified and ligated with HRP insert which had been blunt-ended at the 5’-NdeI site and digested with BamHI. The ligation mixture was used to transform E. coli strain DH5a to ampicillin resistance, and plasmid DNA from several colonies was screened for the presence of HRP insert by digestion with HpaI and BamHI. Plasmids which appeared to contain the HRP gene were then sequenced to check for correct orientation. The plasmid pVLHRP1 has a total size of 10.2 kb and has the HRP gene oriented so that it can be expressed using the baculovirus polyhedrin promoter. Plasmid pVLHRP2, containing the 5’-leader sequence, was constructed by ligating the following three DNA fragments: (a) annealed and kinased synthetic oligonucleotides coding for the cDNA 5’-leader sequence (23) with 5’-PstI and 3’-HpaI restriction sites; (b) HRP insert excised from pUC-HRP with HpaI and BarnHI; and (c) pVL1392 digested with PstI and BamHI (Fig. 1). The ligation mixture was used to transform E. coli strain DH5a to ampicillin resistance, and plasmid DNA from several colonies was screened for the presence of HRP insert by digestion with HpaI and BamHI, or Sal1 and BamHI. Plasmids which appeared to be positive by restriction mapping were then sequenced to ensure that no point mutations had occurred during the transformation and subsequent manipulations. The plasmid pVLHRP2 has a total size of 10.3 kb.

1

2

3

45

6

7

FIG. 4. Western blot of recombinant HRP. Lanes: 1 and 7, molecular weight standards; 2, FPLC-purified recombinant HRP from cell lysate; 3, FPLC-purified recombinant HRP isolated from the medium; 4, St9 cell extract from B-day expression with pVLHRP2; 5, noninfected midlog Sf9 cells: 6, Sigma HRP, Type VI. Approximately 20-40 pg of protein was loaded into each lane.

Production of recombinant virus. Large scale preparations of plasmids pVLHRP1 and pVLHRP2 were purified using Qiagen midi-prep columns. Sf9 cells were coinfected with pVLHRP1 or pVLHRP2 and wild type Autograph californica nuclear polyhedrosis virus (AcMNPV) in the presence of calcium phosphate (27). After 5 days, the medium was removed from the cells and centrifuged. Following three rounds of plaque purification (27), 6 recombinant plaques were isolated from transfection with pVLHRP1 and 25 recombinant plaques were isolated from the pVLHRP2 transfection. The purified viruses were used to infect cell

-

1

2

3

4

5

66

-

45

-

36

-

29 24

-

20.1

6

FIG. 5. SDS-PAGE of recombinant horseradish peroxidase prior to and after incubation with a-mannosidase. Recombinant HRP (36 pg) was incubated with 5 pg a-mannosidase (Lane 3), 0.5 units of N-glycosidase F (Lane 4), or 5 fig a-mannosidase and 0.5 units N-glycosidase F (Lane 5) in 50 mM sodium phosphate buffer, pH 7.0, at 37°C for 28 h. Lanes 1 and 6 are molecular weight standards and Lane 2 is recombinant protein incubated under the same conditions without a-mannosidase or N-glycosidase F. Protein bands at 66 and 45 kDa in Lanes 3 and 5 are from the a-mannosidase preparation. Incubation of the recombinant protein at 37°C also generates minor degradation products that are visible in both the treated and the untreated samples. Approximately lo-20 pg HRP was loaded into each lane.

BACULOVIRUS

EXPRESSION

OF HORSERADISH

65

PEROXIDASE

A

d *b

80

Ppm

FIG. 6. Downfield portion of the 300-MHz proton NMR spectra of ferric heme in the resting state of native (A) and recombinant (B) HRP in 50 mM Na2HP04/sH20 at pH 7.2, 25°C. HRP was allowed to dissolve in 0.5 ml 50 mM Na2HP04/sH20, pH 7.2. Spectra were acquired at 25°C as described under Materials and Methods. Heme resonance assignments were made based upon previously published spectra.

monolayers in 3 ml of Grace’s medium. After 3 days, the cell medium was removed and used as first passage virus stock (pass 1 stock). Fifty microliters of this viral stock was used to infect 1 X lo6 cells in 25mm dishes. Three days later, the cells were harvested, lysed, and analyzed by Western blotting using polyclonal anti-HRP antibodies to determine the presence of recombinant protein. All 6 viruses from pVLHRP1 and 12 of the 25 from pVLHRP2 gave a positive signal in the Western blot. The first passage stock of the virus that yielded the highest level of expression was then used to generate large viral stocks (pass 2 stocks). Two hundred microliters of pass 1 stock was used to infect 1 X 10s cells in a loo-ml culture. After 4 days of growth at 27°C the medium was harvested and used for large scale expression after the titer of the stock had been determined (27). Normally, the titer of this stock was 2-5 X 10’ infectious viruses/ml. Partial purification of HRP from pVLHRP1 recombinant virus. Approximately 200 ml of mid-log St9 cells was centrifuged at 500g and the medium removed. Pass 2 stock was added to the cell pellet to give a multiplicity of infection of 5. After 1.5 h at 27°C the volume was increased to 200 ml in a spinner flask with TNM-FH medium containing 10% FCS and antibiotics. One hour after increasing the volume, 3.5 ml of a 500 pM hemin stock solution in 0.01 N NaOH was mixed with 12.5 ml TNM-FH medium containing 10% FCS. This solution was slowly added to the spinner flask containing the infected cells. The infection was allowed to progress for 40-60 h at 27°C. The cells were harvested by centrifugation at 1000 rpm and were carefully resuspended in 4.5 ml lysis buffer (20 mM NasHPOd, pH 7.2, 100 pM phenylmethylsulfonyl fluoride, 50 gg/ml pepstatin, 20 rc.g/ml leupeptin, and 50 @g/ml aprotinin).

The cells were ruptured by 20 strokes in a dounce homogenizer followed by sonication for 30 s. Cellular debris was removed by centrifugation at 12,000 rpm for 20 min. The supernatant was then centrifuged at 60,000 rpm for 45 min but was found by Western analysis to not contain HRP. The pellet from the low speed centrifugation was then incubated in lysis for 2 h at 4°C. buffer containing 0.5 M NaCl and 1% n-octylglucoside This solution was then centrifuged at 12,000 rpm for 20 min and the supernatant assayed for guaiacol peroxidase activity. The protein was transferred to a polyvinylidene difluoride membrane and subjected to automated Edman degradation using an Applied Biosystems Model 470A protein sequencer at the University of California, San Francisco, Biomolecular Resource Center. Purification of HRP from pVLHRP2 recombinant virus. Infection of St9 cells to produce recombinant HRP which is secreted into the medium was as described for pVLHRP1 with the exception that expression was undertaken in TNM-FH medium containing only 3% FCS and was allowed to continue for 5 days. Purifications of recombinant HRP from both the cell lysate and the medium were performed side by side. Protein from the cell lysate obtained from the ultracentrifugation at 60,000 rpm and from the medium harvested from the initial centrifugation was precipitated at 40 and 90% ammonium sulfate saturation. The 40 and 90% pellets were resuspended in 50 mM NaxHPO,, pH 6.0, and assayed for guaiacol activity. The 90% pellet contained 30-fold higher activity and therefore was chosen for further purification. The dialyzed 90% pellet was chromatographed on 500 ml of Bio-Gel P-100 (Bio-Rad) size exclusion column equilibrated with 50 mM NasHPO+, pH 6.0. The fractions containing peroxidase activity were pooled, concentrated using

66

HARTMANN

30

AND

ORTIZ

25

20

DE MONTELLANO

15

10 wm

FIG. 7. Downfield portion of the high field (500 MHz) proton NMR spectra of ferric low-spin native (A) and recombinant (B) HRP in 50 mM NalHP0J2Hz0 at pH 7.2,25”C. Hyperfine shifted portions of the 500-MHz spectra of HRP-CN at pH 7.2,25”C. Native and recombinant HRPCN samples were generated by addition of 2 eq of KCN to the resting state samples described in Fig. 5. Heme resonance assignments were made based upon the previously published spectra.

an Amicon cell with a YM-30 membrane, and dialyzed against 20 mM sodium acetate, pH 4.3. The protein was then chromatographed by FPLC on a Mono S HR-10 column using a linear gradient of 20 mM sodium acetate, pH 4.3, to 1 M NaC1/20 mM sodium acetate, pH 4.3, over 45 min while monitoring at 280 or 405 nm. Recombinant protein eluted from the column at 109 mM NaCl (Fig. 2). The protein was then dialyzed against 50 mM NazHPO,, pH 6.0, and chromatographed by FPLC on a Mono Q HR-10 column using a linear gradient of 50 mM Na2HPOI, pH 6.0, to 1 M NaC1/50 mM Na,HPO,, pH 6.0, over 45 min while monitoring at 405 nm. HRP does not bind to anion exchange columns at this pH, but there were some impurities present (as determined by 12.5% SDSPAGE) which appeared to elute later in the gradient. Attempts to obtain an N-terminal sequence of the purified protein indicated that, unlike the protein obtained from pVLHRP1, the N-terminus of the secreted HRP was blocked. Recombinant Treatment of recombinant HRP with a-mannosidase. HRP (36 ag) was incubated with either 5 ag of a-mannosidase or 0.5 units of N-glycosidase F, or with both a-mannosidase and N-glycosidase F, for 28 h at 37°C in 50 mM sodium phosphate buffer (pH 7.0). The sample was then analyzed by SDS-PAGE. a-Mannosidase cleaves mannose sugar moieties that have a-linkages to other sugars, while N-glycosidase F cleaves N-linked high mannose, hybrid, and complex oligosaccharides.

RESULTS

AND

DISCUSSION

HRP Expression of HRP in Spodoptera frugiperda. was expressed in baculovirus, a system that has proven quite useful for the high yield expression of eukaryotic

proteins (27), because expression of active HRP in E. coli is confounded by the formation of intractable inclusion bodies (24-26, Hartmann and Ortiz de Montellano, unpublished work). The expression vector was constructed from plasmid pVL1392, which contains a polylinker multiple cloning region downstream of the polyhedrin initiation codon. The synthetic HRP gene, excised from a pUC-19 plasmid by digestion with NdeI, was blunt-ended with T4 polymerase and further digested with BamHI. Plasmid pVL1392 was digested with P&I, blunt-ended with T4 polymerase, and finally digested with BamHI. The synthetic gene was ligated into the vector with T4 ligase. Plasmids containing the HRP insert in the proper orientation were selected by digestion with HpaI and BamHI, or Sal1 and BamHI. In this instance the insert size differed when the gene was present in the correct versus inverse orientation. A single plasmid, pVLHRP1, was chosen from six positive plasmids and was shown to be intact by sequencing from upstream of the insertion point through the 3’-end of the gene. Plasmid pVLHRP1 contained a synthetic gene for HRP but not the 5’-leader sequence found in the plant gene (23). To construct plasmid pVLHRP2, containing the cDNA 5’-leader sequence, the following three DNA frag-

BACULOVIRUS

EXPRESSION

OF HORSERADISH

ments were ligated with T4 ligase: (a) annealed and kinased synthetic oligonucleotides coding for the cDNA 5’leader sequence (23) with 5’-PstI and 3’-HpaI restriction sites; (b) the synthetic HRP insert excised from the pUC19 plasmid with HpaI and BamHI; and (c) pVL1392 digested with PstI and BanHI (Fig. 1). The ligated mixture was used to transform E. coli strain DH5a to ampicillin resistance, and plasmid DNA from several colonies was screened for the presence of the HRP insert by digestion with HpaI and BamHI. Plasmids which appeared to be positive by restriction mapping were sequenced to ensure that no point mutations had occurred during the transformation and subsequent manipulations. To form recombinant viruses containing the HRP insert, Sf9 cells were coinfected with pVLHRP1 or pVLHRP2 and wild type A. californica virus (AcMNPV). Recombinant viruses that had undergone homologous recombination were identified by visual screening of plaque isolates, facilitated by the uptake of trypan blue dye. Putative recombinant plaques were identified by the lack of polyhedrin protein crystals within the cells. Recombinant viruses were isolated and plaque-purified three times to obtain pure recombinant viral stocks. Sf9 cells infected with these viruses were analyzed by SDS-PAGE and Western blotting. All of the recombinant viruses from pVLHRP1 gave positive signals in Western blots at approximately 34 kDa, the anticipated mass of unglycosylated HRP, and 12 of the 25 viruses isolated from

300

400

500

67

PEROXIDASE

pVLHRP2 gave Western blot signals at approximately 43 kDa (not shown). One of each of the recombinant viruses isolated from pVLHRP1 and pVLHRP2 was used to produce large viral stocks. These stocks were used to infect 200-600 ml of Sf9 cells as described under Experimental Procedures. Purification of the protein produced by pVLHRP1 proved to be quite difficult because the expressed protein was soluble only in the presence of detergents. Attempts to purify the protein in the absence of a detergent such as n-octylglucoside or Triton X-100 failed, and the guaiacol activity of the sample did not increase during any phase of the purification. The solubilized protein was subjected to SDS-PAGE on a 12.5% polyacrylamide gel. Eight lanes were transferred to a polyvinylidene difluoride membrane and the 34-kDa band corresponding to the recombinant HRP was excised and subjected to protein sequence analysis. One major sequence was obtained that matched the anticipated first seven amino acids of HRP, including the methionine of the start codon of the synthetic gene (M-Q-L-T-P-T-F). The apparent molecular mass of 34 kDa and localization of the recombinant protein in the cellular debris indicated that the protein was not correctly processed. Therefore, the plasmid pVLHRP2, containing the normal 5’-Nterminal leader sequence, was constructed and used to express recombinant HRP that was secreted into the medium.

600

700

800

WAVELENGTH (nm) FIG. 8. Absorption spectra of compound I of native and recombinant HRP. Approximately an equimolar amount of H202 was added to 86 pg native or 134 pg recombinant HRP (-) in 50 mM sodium phosphate buffer, pH 7.2, to form compound I (- - -). m-visible spectra were recorded immediately upon peroxide addition. Isosbestic points at 360 and 438 nm are similar to literature values for compound I (41). The isosbestic point for the recombinant protein at 355 nm is slightly blue-shifted from the native enzyme due to the presence of a small amount of compound II in the recombinant sample.

68

HARTMANN

AND

ORTIZ

DE MONTELLANO

416 0.12

000

t 250

300

400

600

500

WAVELENGTH

700

600

(nm)

FIG. 9. Absorption spectra of compound II of native and recombinant HRP. Approximately 2 eq of H202 were added to 17 pg native (-) or 25 Fg recombinant HRP (-*-a ) in 50 mM sodium phosphate buffer, pH 7.2, to form compound II. uv-visible spectra were recorded 2-3 min after addition of peroxide. The difference in absorbance at 280 nm is due to the presence of polyethylene glycol in the recombinant sample.

Purification of recombinant HRP from virus pVLHRP2. Mid-log St9 cells (2 X lo6 cells/ml) were infected at a multiplicity of infection of 5. Five days after infection, the cells were removed from the medium by centrifugation, allowing purification of HRP from the medium. Recombinant protein was purified from both the cells and the medium only if the time of expression was 2-3 days. Excessive cell lysis caused by advanced viral infection precluded recovery of the protein from infected cells after 3 days. Recombinant HRP was rapidly purified loo-fold from the medium by ammonium sulfate precipitation, size exclusion chromatography, cation exchange, and finally anion exchange chromatography (Fig. 2), yielding 4 mg of pure recombinant HRP/l Sf9 cells (Table I; Figs. 3 and 4). Expression of the gene in Sf21 rather than Sf9 cells for 8 days yields, after purification, 20 mg/liter of the enzyme. It is likely that this yield can be substantially improved by optimizing the culture and purification conditions. Physical properties of recombinant HRP. Based on comparison of the mobility of the recombinant protein with standard markers and native HRP, the molecular weight of the majority of the recombinant protein is 43,000 (Fig. 3). The N-terminus of the active protein from pVLHRP2 appears to be blocked and, unlike that from pVLHRP1, could not be sequenced. A blocked N-terminus in the processed protein is consistent with the fact that the N-terminus is blocked in HRP isolated from horseradish root (22). Baculovirus is able to N-terminally block proteins because chloramphenicol acetyltransferase and

tyrosine hydroxylase are N-terminally blocked when expressed in baculovirus (31,32). The protein purified from the medium does not form a discreet band on the gel (lane 3), indicating a heterogeneous protein population due (probably) to differing levels of glycosylation. The protein isolated from the cell lysate (lane 2) is also diffuse but exhibits two distinct bands at approximately 50 and 47 kDa. Both mammalian and insect cells initially transfer high mannose oligosaccharide precursors to asparagine residues in the polypeptide chain. However, mammalian cells then trim the high mannose structure to a core oligosaccharide to which they add a variety of terminal sugar residues (33). Insect cells do not add terminal sugars after trimming the high mannose structure. The protein from the cell lysate clearly has not been fully processed with respect to trimming of the carbohydrate side chains. The differences in N-linked oligosaccharide structure do not appear to alter the catalytic function with respect to that of native HRP (vide infra). Reconstitution of the purified enzyme with hemin dissolved in 0.01 N NaOH does not affect the observed R, value, indicating full incorporation of heme during expression. Western blot analysis of SDSPAGE gels (Fig. 4) confirms the presence of differentially processed forms of recombinant HRP in the purified sample, none of which are present in uninfected Sf9 cells. Treatment of recombinant HRP with a-mannosidose. Western blot analysis and SDS-PAGE of the purified recombinant protein (Figs. 3 and 4) indicate a heterogeneous protein population. This heterogeneity can be decreased by incubating the recombinant enzyme with LY-

BACULOVIRUS

EXPRESSION

OF HORSERADISH

69

PEROXIDASE

545

250

300

400

500

WAVELENGTH

600

700

600

(nm)

a loo-fold excess of H202 was added to native FIG. 10. Absorption spectra of compound III of native and recombinant HRP. Approximately (17 ag) or recombinant (25 ng) HRP in 50 mM sodium phosphate buffer, pH 7.2, to form compound III. uv-visible spectra were recorded 4-6 min after addition of peroxide.

mannosidase, which catalyzes the trimming of high mannose N-linked complex oligosaccharides. SDS-PAGE analysis of recombinant HRP incubated 28 h at 37°C with either a-mannosidase or N-glycosidase F, or with both (Ymannosidase and N-glycosidase F, shows that a-mannosidase decreases the apparent molecular weight of the recombinant protein by approximately 3000 Da and tightens the protein band (Fig. 5, Lanes 3 and 5). N-Glycosidase F does not detectably alter the recombinant protein. Detailed studies of the carbohydrate content of the recombinant enzyme and of the homogeneity of the protein sample generated by cr-mannosidase are in progress. It is clear, however, that a-mannosidase can be added to purified, recombinant HRP to generate a protein that is more homogeneous and approximately 3-5 kDa lower in molecular weight. Treatment of the recombinant enzyme with a-mannosidase does not detectably alter its catalytic activity. NMR characterization of recombinant HRP. The NMR spectra of the resting state (high spin) and cyanide complex (low spin) of 3.7 mg of recombinant HRP were compared to the equivalent spectra of the native protein (Figs. 6 and 7). The resolvable regions of the NMR spectra

of the native and recombinant proteins are essentially identical. Specifically, based on previous assignment of the heme resonances within the active site (11, 34-37), the environments of the four methyls, vinyl substituents, propionic acid a-protons, and the 4-proton of the proximal histidine are the same in the native and recombinant enzymes. This is strong evidence that the active site is intact because NMR readily detects differences in the environments of these groups in, for example, HRP isozyme A (Dr. Gerd La Mar, personal communication). Catalytic intermediates and catalytic activity. The heme content of the protein, as determined from the intensity of the Soret absorbance at 402 nm relative to the protein absorption at 275 nm (Table I), is that expected for HRP with a fully occupied prosthetic heme crevice. Furthermore, activity measurements using guaiacol as a substrate indicate that the recombinant protein has essentially the same catalytic activity (19.6 ~mol/min/nmol) as native HRP (21.3 pmol/min/nmol) when its concentration is estimated from the Soret absorbance. To further characterize the catalytic activity of the enzyme, we have determined the spectra of the compound I and II intermediates formed by reaction with H202 (Figs. 8 and 9).

70

HARTMANN

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DE MONTELLANO

0.40

0.32

250

300

400

500

600

700

600

900

WAVELENGTH (nm) FIG. 11. uv-visible absorption spectra of the time course for isoporphyrin formation at 847 nm. (A) 3.43 pM native HRP in 50 mM sodium phosphate buffer, pH 7.2; 0.343 mM ethylhydrazine; and 0.343 mM HrOr, reacted until the enzyme was >90% inactivated. The reaction mixture was then passed down a Sephadex G-25 column to remove excess ethylhydrazine and Hz02. Spectra were then collected every 5 min. (B) Recombinant HRP: same reaction conditions as in A. As described by Ator et al. (28,29), there is an observed shift of the Soret from 404 to 417 nm, concomitant with an increase in absorbance at 847 nm. The increase and subsequent decay at 847 nm have been attributed to a putative isoporphyrin intermediate. Guaiacol activities were identical in both samples before and after inhibition by ethylhydrazine.

In both cases, the spectra are nearly identical to those obtained with the native enzyme. The third standard redox state of HRP is compound III, analogous to that of oxymyoglobin, in which molecular oxygen is bound to the ferrous prosthetic group (1, 2). As shown in Fig. 10, the spectrum of compound III is essentially the same for the recombinant and native enzymes. Chemical evaluation of active site integrity. Recent work has shown that HRP reacts with ethylhydrazine to give an isoporphyrin intermediate that gradually decays to generate a heme group specifically substituted with an ethyl group at the d-meso position (29, 30). Reaction of recombinant HRP with ethylhydrazine gives the same isoporphyrin spectrum observed with native HRP (Fig. ll), and isolation of the prosthetic group after decay of the isoporphyrin yields exclusively B-meso-ethylheme (not shown). This provides strong evidence for the integrity of the active site of the recombinant protein. First, addition of the ethyl radical only to the d-meso position, when there are three other possible meso positions, argues

that the general topology of the active site is the same in the recombinant and native enzymes. Second, and even more important, isoporphyrins with one substituent and one hydrogen at the affected meso carbon are so unstable that they cannot be detected in solution. The positively charged isoporphyrin is therefore stabilized by still undefined but probably electrostatic interactions with the protein (30). Formation of the stable isoporphyrin requires that the active site of the recombinant enzyme preserve the relatively subtle structural features that result in isoporphyrin stabilization. of thioanisole and para-methoxythioaniOxidation sole. The sulfoxidation of thioanisoles by HRP differs from other HRP-catalyzed reactions in that the ferry1 oxygen of compound I appears to be transferred directly to the sulfur (38-40). Kinetic studies showing that compound II is an intermediate in the reaction suggest the sulfur radical cation is formed by a conventional oneelectron abstraction, but subsequently combines with the ferry1 oxygen rather than diffusing out of the active site

BACULOVIRUS

EXPRESSION

OF HORSERADISH

71

PEROXIDASE

0.4

0.3

Y f

0.2,

I? 8 $

0.11

0.0

0.l

250

300

500

400

FIG.

II

Stereoselectivity of Sulfoxide Formation in the Reaction of Native and Recombinant HRP with Thioanisole and para-Methoxythioanisole” Substrate/enzyme Thioanisole Native HRP Recombinant HRP para-Methoxythioanisole Native HRP Recombinant HRP

S-(-):R-(+)

ratio

600

900

(nm)

1 l-Continued

(41,42). We have recently found, contrary to two reports that the sulfoxidation occurs without enantioselectivity (39,43), that the reaction is actually quite stereoselective (R. Z. Harris and P. R. Ortiz de Montellano, unpublished). Thus, by decreasing the reaction time from days to 1 h, we have observed enantiomeric excesses of approximately

TABLE

700

600

WAVELENGTH

ee (%)

2.5:l.O

42.9

2.4:l.O

41.2

5.4:l.O 4.O:l.O

68.8 60.0

a Reaction conditions were typically 10 HIM enzyme, 5 mM thioanisole substrate, 1 mM H,Os, 50 mM sodium phosphate buffer, pH 7.2, 25°C. Sulfoxide products were isolated by HPLC on a Daicel OB chiral column under isocratic conditions (80:20 hexane:isopropanol), flow rate = 0.5 ml/min.

41 and 60% for thioanisole andpara-methoxythioanisole oxidation, respectively (Table II). The thioanisole and the S-(-) and R-(+) sulfoxidation products, readily separated by HPLC on a Daicel OB chiral column, eluted at 12.5, 24.5, and 38.1 min, respectively. It is likely that the previously reported lack of stereoselectivity was an artifact of the long incubation times that were employed (39; 43). Control experiments show that detectable sulfoxidation occurs with HzOz alone within 1 h. The small amount of racemic product obtained in control incubations was subtracted from that obtained with HRP before the enantiomeric excesses were calculated. The stereospecificity of the sulfoxidation reaction provides a demanding test of the integrity of the substrate binding site. It is therefore important that very similar enantiomeric excesses are found when the sulfoxidation is catalyzed by the native and recombinant enzymes (Table II). CONCLUSIONS A synthetic HRP gene, including the V-leader sequence, has been expressed in good yield in S. frugiperda cells. The expressed enzyme is terminally blocked and glycosylated. Spectroscopic and functional criteria indicate that the active sites of the recombinant and native enzymes

72

HARTMANN

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are identical despite probable differences in glycosylation patterns. Thus, the NMR spectra and the absorption spectra of compounds I, II, and III formed by reaction of the native and recombinant enzymes with HzOz are virtually identical. Catalytically, the recombinant enzyme exhibits the same guaiacol oxidizing activity as the native enzyme, oxidizes thioanisole to the sulfoxide with comparable enantioselectivity, and reacts with ethylhydrazine to give a similar protein-stabilized isoporphyrin that decays exclusively to d-meso-ethylheme. Successful expression of HRP in good yields makes possible investigation of its structure and function by site specific mutagenesis and facilitates the production of protein suitable for Xray crystallography. ACKNOWLEDGMENTS We sincerely thank Dr. Janos Taljanidisz for his help with general cloning techniques, Dr. David Morgan for his advice on baculovirus expression, Drs. Gerd La Mar and Jeff de Ropp for invaluable help in obtaining the NMR data, and Ms. Krista Timlin for help with the figures and manuscript.

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