Vol. 179, No. 2, 1991 September 16, 1991
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 897-903
HETEROLOGOUS EXPRESSION OF ACTIVE MANGANESE PEROXIDASE FROM PHANEROCHAETE CHRYSOSPORIUM USING THE BACULOVIRUS EXPRESSION SYSTEM Elizabeth A. Pease*,Steven D. A&
and Ming Tien*§
*Departmentof Molecular and Cell Biology, The PennsylvaniaState University University Park, PA 16802 SBiotechnology Center, Utah State University, Logan, Utah 84322 Received
July
29,
1991
The cDNA encodingMn peroxidaseisozyme H4 from Phanerochaerechrysosporiumwas recombinedinto a baculovirus and heterologouslyexpressedin Sf9 cells. The recombinantMn peroxidasehasthe samemolecular weight asthe native enzyme asdeterminedby SDS-PAGE and cross-reactswith a Mn peroxidase-specificantibody. The recombinantenzyme hasa slightly lower p1 than the native fungal isozyme H4 indicating somedifferencesin post-translationalmodification. Phenolred, guaiacol, and vanillylacetone, substratesof the native Mn peroxidase,are oxidized by the recombinantenzyme. All of the activities aredependenton both Mn (II) and Hz@. 0 1991 Academic Przess, Inc. We report herethe heterologousexpressionof active Mn peroxidase(isozyme H4) using the baculovirus expressionsystem. At leastten hemeproteins,thought to be involved in lignin degradation,have beenidentified in the extracellular fluid of ligninolytic culturesof Phanerochaete chlysosporiwn (l-3). Theseenzymescan be classifiedinto two catalytically distinct types of peroxidases,the lignin peroxidases(ligninases)(4,5) and the Mn peroxidases(6,7). The former peroxidasesdirectly oxidize nonphenolicsubstrates(8) whereasthe latter oxidize phenolic substratesusing Mn asa mediator (9). The enzyme first oxidizes Mn (II) to Mn (III), which in turn, oxidizes the phenolic substrates. The cDNAs encodingmany of the lignin (10,11,12) and Mn peroxidases(13,14) isozymes have beenisolated. With thesecDNAs, many industrial andacademiclaboratorieshave attempted heterologousexpressionof active lignin and Mn peroxidase. To date, we are not awareof any reportsdemonstratingexpressionof active recombinantenzyme. Expressionin prokaryotic systemshasresultedin production of inclusion bodiescontaining inactive protein without the heme inserted(15). The enzymeshave beendifficult to reconstitute(15). Many eukaryotic proteins have beensuccessfullyproducedin the baculovirusexpressionsystem(16,17). Mammalian cytochrome P450, a hemecontaining protein, hasbeenactively expressedin this system(18). The expressionof anotherhemeprotein,lignin peroxidaseisozyme H8 in baculovirus wasrecently reported (19). Descibedhere is the expressionand characterizationof active recombinantMn peroxidase. &To whom correspondenceshould be addressed. 0006-291X/91 $1.50 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
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Materials Hemin was purchased from Porphyrin Products (Logan, UT); tissue culture cell factories from Nunc; Grace’s medium from Gibco; fetal calf serum from Sigma Chemical Co. 39 cell Spodopterafnrgiperda were generously supplied by Max Summers (Texas A&M). The pEVmod plasmid and vETLPga1 Autographa californica nuclear polyhedrosis virus were generously supplied by Lois Miller (University of Georgia). Construction of transplacement vector The 1.3 kb cDNA hMP-1 insert (13) was cloned into the EcoRI site of the pEVmod vector with the polyhedrin promoter region 5’ to the cDNA. A large-scale preparation was CsCl purified and used as the transplacement vector to recombine the LMP- 1 cDNA into the baculovirus vETLpga1. Selection of recombinant virus Procedures for cotransfection, screening and maintenance of Sf9 cells were as described by Summers and Smith (20). The virus vETLPga1 was used to facilitate screening of recombinants since it contains the &galactosidase gene (21) which caused all infected cells to be blue when grown in the presence of X-gal (5-bromo-4-chloro-3-indolyl-P-Dgalactoside). Positive clones were first identified as blue occlusion-negative plaques and then confiied by performing Western slot blots on the cell lysates (data not shown). Three rounds of purification were needed to purify the virus. Production of recombinant Mn peroxidase The recombinant protein was produced in either 2or IO-level cell factories using 0.2 or 1.O L of medium, respectively. Cell factories were seeded to approximately 50% confluency. At 95% confluency, the medium was removed and the cells were infected with the pEVH4 recombinant virus at a multiplicity of infection of 10 plaque forming units per cell. The virus was attached in 100 ml or 500 ml of 1X Grace’s complete, 10% fetal calf serum, and antibiotics for 1 hour and then an equal voiume of the same medium was added. These were incubated at 27°C for 60 hours at which time the medium was removed (concentrated 20 fold, and saved for further analysis) and replaced with fresh serum free and additive free 1X Grace’s with the addition of hemin to a final concentration of 1 ~.&ml. Hemin was dissolved in 50 mM NaOH at a concentration of 1 mg/ml, filter sterilized and added to the medium before addition to the cell factory. These were incubated for an additional 12-14 hours at which time the medium was removed for Mn peroxidase purification. Partial purljkation of recombinant Mn peroxidase The supematant from virus-infected St9 cells was centrifuged (4000 RPM) at 4’C for 10 minutes. The supematant was then concentrated 20 fold (Amicon YMlO membrane) and dialyzed against 5 mM sodium succinate, pH 6.5. This was loaded onto a 100~ml DEAE Biogel A column and eluted with a O-O.5 M NaCl gradient. The active fractions were dialyzed against 5 mM sodium succinate pH 6.5 for storage. Isoelectric focusing Isoelectric focusing (IEF) gels were run using a Pharmacia LKB Multiphor II electrophoresis system. Gels of 5% acrylamide were made by mixing 0.9 ml of pH 4-6 ampholine and 0.6 ml of pH 3.5-5.0 ampholine in a total volume of 30 ml. Mn peroxidase activity was visualized in IEF gels by a modified method of Kersten and Kirk (22). The gel was submerged 200 ml of 82.5 mM sodium succinate, pH 4.5, 82.5 mM sodium lactate, pH 4.5,4.95 mg/ml gelatin, 0,165 mM MnS04 and 0.48 mM phenol red. The color reaction was initiated by the addition of 1 ml of 10 mM H202. After 10 minutes, a second aliquot of H202 was added and the color allowed to develop further. Western blot visualization of the IEF gels was performed by fixing the gels in 0.16 M sulphosalicylic acid, 0.7 M trichloroacetic acid for 30 minutes. The gel was then soaked for 30 minutes in three changes of 12.3 mM Tris HCl, 0.096 M glycine pH 8.6 with 10% methanol and 0.001% SDS at 50°C. The remainder of the procedure was the same as that for an SDS-PAGE Western transfer. Enzyme Activity Assays One unit is defined as one l.trnol of product formed per minute, Mn peroxidase activity was measured by phenol red oxidation except where noted otherwise. The procedure is as described by Glenn and Gold (9) with the exception that a 5.5 ml reaction mix was used and 1 ml was removed every minute and added to 40 pl of 5 M NaOH. Oxidized phenol red was measured at ODelo using an extinction coefficient of 22.0 mM-l . cm-l (23). Vanillylacetone oxidation was assayed by substrate disappearance at 336 nm using an extinction coefficient of 18.6 m~-l. cm-l (24). Guaiacol oxidation was assayed as previously described (25) by measuring tetraguaiacol formation at 465 nm using an extinction coefficient of 26.6 mM-l. cm-l (26). SDS-PAGE gels and Western transfer SDS-PAGE was performed as described by Laemmli (27). Western blots were performed using a polyclonal antibody specific for H4 and the anti-rabbit alkaline phosphatase conjugate. 898
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Results Production of recombinantMnperoxiduse in $9 cells Infection of Sf9 cells with the recombinant&4P-l-containing virus resultedin expressionof active Mn peroxidase. As with the native fungus, the recombinantMn peroxidasewas found in the extracellular medium. At 60 hours post infection, the medium waschangedto 1X Grace’s(serumand additive free) containing hemin at a final concentrationof 1 @ml. Changingthe mediumincreasedenzyme yield and simplified enzyme purification. The mediumwasharvestedat 74 hourspost infection. The activity of the supematant(1 liter) wasmeasuredat 35.7 U/L. Although the addition of hemin elevatedactivity, a low level of activty (lessthan 2 U/L) wasdetectedbefore the mediumchangeand addition of hemin. The enzyme waspurified on a DEAE Biogel-A column for further analyses. Properties of recombinantMnperoxidase The Mr of the recombinantH4, determinedby SDSPAGE, is identical to the native fungal Mn peroxidase. Fig. 1 showsa Western blot of a SDSPAGE gel where the Mn peroxidasewasvisualized using an anti-H4 antibody. The blot showsa singleimmunoreactivebandin the concentratedmediumof infected cells. No immuno-reactive bandswere found in the mediumfrom uninfected Sf9 cells (CC) or vETLP-gal infected cells (VC) (Fig. 1). Purified fungal Mn peroxidaseisozymesH4, H.5, and H3 were run asstandardson the gel.
B P
VC
rMP
nH4
nH5
nH3
rMP
rMP
C
nMP
rMP
nMP
nMP
CC
02
;a Gx
+
+
Fig. 1. SDS-PAGE of recombinant Mn peroxidase (rMP) andnativeMn peroxidase (nMP). Samples weresubjected to SDS-PAGEandvisualizedby Westernblottingtechniques. VC: virus controlcontaining40 pJof supematant from nonrecombinant virus-infectedSf9cells; nH4: 1.8Kgof nativeisozymeH4, rMl? 5 pl of 20-foldconcentrated recombinant supematant; nH5: 1 kg of nativeH5; nH3: 0.8 Fg of nativeH3; andCC: cell controlcontaining40 1.11 of
supematant from non-infectedSt9 cells. Fu Isoelectricfocusinganalyses of recombinant andnativeMn peroxidase.Thecrude recombinant Mn peroxidase(rMP) andthepurifiednativeMn peroxidase (nMP) weresubjected to IEF asdescribedin MaterialsandMethods.Theanode(+) was1M H3PO4 andthecathode(-) was1M NaOH. Samples werevisualizedby Coomassie bluestaining(A), Westernblotting(B), or activity stainingwith phenolred(C). PanelA contained10pl of a 20-foldconcentrated supernatant from recombinant H4 virus-infectedSB cells(rMP) and3.5Kg of purifiednativeH4 from P. chrysosporium(nMP). PanelsB andC contained2 ~1of rMP and0.7 pg of nMP.
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Minutes &gJ Time course of phenol red oxidation catalyzed by recombinant Mn peroxidase. Incubations contained 1 ml of crude recombinant supematant in a total of 5.5 ml. Aliquots were removed and assayed for oxidized phenol red as described in Materials and Methods. Incubations contained all of the necessary cofactors (0), minus MnS04 (O), or minus Hz02 (A).
Fig. 2A showsthe total proteinsof the extracellular mediumseparatedby IEF and visualized by Coomassieblue staining. A bandfocusedto approximately the samepI asnative isozyme H4 (~14.6). Western transfer analysisindicated that a bandwith slightly lower p1cross-reactswith an anti-H4-specific antibody (Fig. 2B). The IEF gel, when stainedfor Mn peroxidaseactivity, revealed an intensebandcorrespondingto the immuno-reactiveband(Fig. 2C). There are two minor bandswith phenolred oxidizing activity which are not associatedwith the insect tissue culture. Thesebandsmay be due to partially degradedrecombinantH4 or H4 which has undergonedifferent processing. Substrateprofile of Mn peroxidase Fig. 3 showsthe time coursefor phenol red oxidation by the cruderecombinantMn peroxidasepreparation. Similar to native Mn peroxidase(7), activity is dependenton the presenceof addedMn (II) andHz02 andis linear over time. Phenolred, guaiacol andvanillylacetone, all substratesof the native H4 Mn peroxidasefrom P. chrysosporium (25), are alsooxidized by the recombinantMn peroxidase.Theseactivities are all Mn (II) and Hz& dependentasshown in Table 1.
Discussion We report here the heterologousexpressionof active recombinantMn peroxidasefrom the white-rot fungus P. chrysosporium. The cDNA IMP-l,
previously
identified
Table 1. Activity profile of recombinant Mn peroxidase” Complete minus H202 minus
Mn
(II)
Phenol Red 83 0.8
Guaiacol
Vanillvlacetone
94 0.0
201 1.2
0.0
0.0
5.0
cActivities were determined as per Materials and Methods and am reported in units/L x 1000. The complete reaction mixture contained all the cofactors as described in Materials and Methods. Incubations contained 200 pl of partially purified recombinant Mn peroxidase (rH4).
to encode Mn
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(13), was recombined into the baculovirus
vETLPgal and heterologously expressed in St9 cells. The active recombinant enzyme is secreted into the extracellular medium. The baculovirus expression system has proven to be an excellent technique for expression of eukaryotic enzymes (17,lQ It is especially applicable when various types of post-translational modification are required for enzyme activity (28). This was demonstrated here with the Mn peroxidase. The native H4 undergoes three types of post-translational modification when expressed in P. chrysosporium: i) heme insertion, the Mn peroxidases contain one protoporphyrin IX per enzyme molecule (29); ii) proteolytic processing, the cDNA AMP-1 encodes a leader sequence which directs the enzyme for secretion and is removed (13); and iii) glycosylation, the Mn peroxidases are glycosylated (25). Although we have not purified and characterized the recombinant H4 for the modifications mentioned above, our results suggest that all three are occurring with the baculovirus system. The spectral properties of the recombinant enzyme suggest that heme is inserted. Enzyme activity is associated with a 406 nm-absorbing species (Soret). Furthermore, because heme is part of the active site, it is highly unlikely that activity can be attributed to apoenzyme. Evidence for proper processing of the leader sequence comes from two findings. The first and most direct evidence is that the recombinant enzyme is found in the extracellular medium. The second piece of evidence is that the apparent Mr of the recombinant enzyme, as determined by SDS-PAGE is identical to that of native H4. If the leader sequence was not removed, the Mr would increase approximately 3 kd. Secretion of heterologous proteins using the native leader sequence has been previously observed with the baculovirus expression system and therefore is not a surprising finding here for Mn peroxidase (16,28,30). However, unequivocal demonstation of proper processing of the leader sequences would require purification of the recombinant enzyme and N-terminal amino acid sequencing. The Mn peroxidase isozyme H4 amino acid sequence contains four potential N-linked glycosylation sites (Asn-X-Thr/Ser)
as well as numerous O-linked glycosylation sites (13). Even
though insect cells have been reported to utilize the same N-glycosylation
sites as most vertabrates
and yeast (31), they do not contain sialic acid and practically no galactosyl and sialyl transferases (32). This results in proteins expressed in insect cells having the same core glycosylation, but no complex carbohydrate moieties (31). Little is known about O-linked glycosylation in insect cells (33). The binding of recombinant H4 to a Concanavalin A column suggests that the enzyme is glycosylated (data not shown).
Concanavalin A binds to oligosaccharides containing terminal
mannose residues (34). The only noticeable difference between the recombinant and native enzyme is the p1. The p1 difference would indicate that the processing of the recombinant Mn peroxidase is not identical to that of native Mn peroxidase. The slight differences may be due to a variation in the glycosylation. Nevertheless, the recombinant Mn peroxidase oxidizes the same substrates as the native enzyme (phenol red, guaiacol, and vanillylacetone) All activities are dependent on Mn (II) and Hz% as with the native protein. The relative amount of activity for each substrate is the same for the recombinant and the native Mn peroxidase. Determining whether the recombinant enzyme exhibits similar kinetic parameters of bat and k&& awaits further purification of the enzyme. 901
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Although not essential for production of active enzyme, the addition of hemin to the medium increased activity by at least 15 fold. A similar result was also observed for the expression of mammalian cytochrome P450 in the baculovirus system (18). The addition of hemin to the medium increased the P450 content five to six fold at 72 hours post infection. Addition of hemin to the growth medium at 60 hours post infection correlates with the time of peak recombinant protein production (18). The level of Mn peroxidase activity produced in the baculovirus system is comparable to that produced by the fungus, Although scale up of the baculovims is easier than the fungus, the baculovirus expression system is relatively expensive. Therefore, it will probably not replace the fungus for large-scale enzyme production. However, the baculovirus system will be valuable for mechanistic studies. It will provide purified enzyme free of any possible contamination from other isozymes. There are over ten extracellular lignin-degrading peroxidases produced in P. chysosporim.
Assignment of an activity to a specific isozyme isolated may be problematic due to
possible impurities if the enzyme is isolated from the fungus. The expression of active Mn peroxidase will also permit structure/ function studies using the tools of site-direct mutagenesis.
Acknowledgments This work was supported in part by United States Department of Energy Grant DE-FGOZ 87ER136990 and National Institute of Environmental Health Sciences Grant l-P42ESO4922-01. Ming Tien is a recipient of a Presidential Young Investigator Award from the National Science Foundation (DCB-8657853).
We thank Todd Johnson for his help in transfecting and purifying
the recombinant virus.
References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
Kirk, T. K. and Farrell, R.L. (1987) Ann. Rev. Microbial. 41,465-505. Kirk, T. K., Croan, S., Tien, M., Murtagh, K. E., and Farrell, R. L. (1986) Enzyme Microb. Technol. 8,27-32. Farrell, R. A., Murtagh, K., Tien, M., Mozuch, M. D., and Kirk, T. K. (1989) Enz. Microbial. Tech. 11, 322-328. Tien, M. and Kirk, T. K. (1983) Science 221, 661-663. Glenn, J. K., Morgan, M. A., Mayfield, M. B., Kuwahara, M., and Gold, M. H. (1983) Biochem. Biophys. Res. Commun. 114, 1077-1083. Paszczynski, A., Huynh, V.-B., and Crawford, R. (1985) FEMS Microbial. Lett., 28, 119128. Kuwahara, M., Glenn, J. K., Morgan, M. A., Gold, M. H. (1984), FEBS Lett., 169, 247249. Kirk, T. K., Tien, M., Kersten, P. J., Mozuch, M. D., and Kalyanaraman, B. (1986) Biochem. J. 236,279-287. Glenn, J. K. and Gold, M. H. (1985) Arch. Biochem. Biophys. 242, 329-341. Tien, M. and Tu, C.-P. D. (1987) Nature 326,520-523; also Nature 328,742. de Boer, H. A., Zhang, Y. Z., Collins, C., and Reddy, C. A. (1987) Gene 60,93-102. Andrawis, A., Pease, E. A., Kuan, I., Holzbaur, E., and Tien, M. (1989) B&hem. Biophys. Res. Comm. 162, 673-680. Pease, E. A., Andrawis, A., and Tien, M. (1989) J. Biol. Chem. 264, 13531-13535. Pribnow, D., Mayfield, M. B., Nipper, V. J., Brown, J. A., Gold, M. (1989), J. Biol. Chem. 264,5036-5040. Andrawis, A., Pease, E. and Tien, M. (1990) in Biotechnology in Pulp and Paper Manufacture Applications and Fundamental Investigations (T. K. Kirk and H-m. Chang, ed.) Butterworth-Heinemann., 601-613, Butteworth-Heinemann, Boston, 902
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16. Smith, G. E., Summers,M. D., Fraser, M. J. (1983) Molec. Cell. Biol. 3,2 156-2165. 17. Miller, L. K. (1988) Ann. Rev. Microbial. 42, 177-199. 18. Asseffa, A., Smith, S. J., Nagata, K., Gillette, J., Gelboin, H. V., and Gonzalez, F. J., (1989) Arch. Biochem. Biophys. 274,481-490. 19. Johnson,T. M. and Li, J. K.-K. (1991) FASEB Journal 5, A1546. 20. Summers,M. D., and Smith, G. E. (1987) Tex. Agric. Exp. Stn. Bull. No. 155. 21. Crawford, A. M. amd Miller, L. K. (1988) J. Virology 62,2773-2781. 22. Kersten, P. J., and Kirk, T. K. (1987) J. Bacteriology 169,2195-2201. 23. Pick, E. and Keisari, Y. (1980) J. of Immunological Methods 38, 161-170. 24. Paszczynski, A., Huynh, V.-B., and Crawford, R. (1985) FEMS Microbial. Lett. 29,37-41. 25. Paszczynski, A., Huynh, V.-B., and Crawford, R. (1986) Arch. Biochem. Biophys. 244, 750-765. 26. Chance,B. and Maehly, A. C. (1955) Methods in Enzymology 2,764-775. 27. Laemmli, U. K. (1970) Nature 227,680-685. 28. Miller, D. W., Safer, P., and Miller, L. K. (1986) in Genetic Engineering (J. K. Setlow, A. Hollaender, Ed.) 8: 277-298. Plenum, New York. 29. Mino, Y., Wariishi, H., Blackburn, N., J., Loehr, T., M., and Gold, M. H. (1988) J. Biol. Chem. 263,7029-7036. 30. Maeda,S., Kawai, T., Obinata, M., Fujiwara, H., Horiuchi, T., Saeki, Y., Sato, Y., and Furasawa,M. (1985) Nature 315, 592-594. 31. Hsieh, P. and Robbins, P. W. (1984) J. Biol. Chem. 259, 2375-2382. 32. Butters, T.D., Hughs, R.C. and Vischer, P. (1981) Biochem. Biophys. Acta 640, 672-686. 33. Butters, T.D., Hughs, R.C. (1981) Biochem. Biophys. Acta 640, 655-671. 34. Sharon, N. and Lis, H. (1972) Science 177,949-952.
903
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 897-903
HETEROLOGOUS EXPRESSION OF ACTIVE MANGANESE PEROXIDASE FROM PHANEROCHAETE CHRYSOSPORIUM USING THE BACULOVIRUS EXPRESSION SYSTEM Elizabeth A. Pease*,Steven D. A&
and Ming Tien*§
*Departmentof Molecular and Cell Biology, The PennsylvaniaState University University Park, PA 16802 SBiotechnology Center, Utah State University, Logan, Utah 84322 Received
July
29,
1991
The cDNA encodingMn peroxidaseisozyme H4 from Phanerochaerechrysosporiumwas recombinedinto a baculovirus and heterologouslyexpressedin Sf9 cells. The recombinantMn peroxidasehasthe samemolecular weight asthe native enzyme asdeterminedby SDS-PAGE and cross-reactswith a Mn peroxidase-specificantibody. The recombinantenzyme hasa slightly lower p1 than the native fungal isozyme H4 indicating somedifferencesin post-translationalmodification. Phenolred, guaiacol, and vanillylacetone, substratesof the native Mn peroxidase,are oxidized by the recombinantenzyme. All of the activities aredependenton both Mn (II) and Hz@. 0 1991 Academic Przess, Inc. We report herethe heterologousexpressionof active Mn peroxidase(isozyme H4) using the baculovirus expressionsystem. At leastten hemeproteins,thought to be involved in lignin degradation,have beenidentified in the extracellular fluid of ligninolytic culturesof Phanerochaete chlysosporiwn (l-3). Theseenzymescan be classifiedinto two catalytically distinct types of peroxidases,the lignin peroxidases(ligninases)(4,5) and the Mn peroxidases(6,7). The former peroxidasesdirectly oxidize nonphenolicsubstrates(8) whereasthe latter oxidize phenolic substratesusing Mn asa mediator (9). The enzyme first oxidizes Mn (II) to Mn (III), which in turn, oxidizes the phenolic substrates. The cDNAs encodingmany of the lignin (10,11,12) and Mn peroxidases(13,14) isozymes have beenisolated. With thesecDNAs, many industrial andacademiclaboratorieshave attempted heterologousexpressionof active lignin and Mn peroxidase. To date, we are not awareof any reportsdemonstratingexpressionof active recombinantenzyme. Expressionin prokaryotic systemshasresultedin production of inclusion bodiescontaining inactive protein without the heme inserted(15). The enzymeshave beendifficult to reconstitute(15). Many eukaryotic proteins have beensuccessfullyproducedin the baculovirusexpressionsystem(16,17). Mammalian cytochrome P450, a hemecontaining protein, hasbeenactively expressedin this system(18). The expressionof anotherhemeprotein,lignin peroxidaseisozyme H8 in baculovirus wasrecently reported (19). Descibedhere is the expressionand characterizationof active recombinantMn peroxidase. &To whom correspondenceshould be addressed. 0006-291X/91 $1.50 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
897
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179,
Materials
No.
2, 1991
BIOCHEMICAL
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RESEARCH
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and Methods
Materials Hemin was purchased from Porphyrin Products (Logan, UT); tissue culture cell factories from Nunc; Grace’s medium from Gibco; fetal calf serum from Sigma Chemical Co. 39 cell Spodopterafnrgiperda were generously supplied by Max Summers (Texas A&M). The pEVmod plasmid and vETLPga1 Autographa californica nuclear polyhedrosis virus were generously supplied by Lois Miller (University of Georgia). Construction of transplacement vector The 1.3 kb cDNA hMP-1 insert (13) was cloned into the EcoRI site of the pEVmod vector with the polyhedrin promoter region 5’ to the cDNA. A large-scale preparation was CsCl purified and used as the transplacement vector to recombine the LMP- 1 cDNA into the baculovirus vETLpga1. Selection of recombinant virus Procedures for cotransfection, screening and maintenance of Sf9 cells were as described by Summers and Smith (20). The virus vETLPga1 was used to facilitate screening of recombinants since it contains the &galactosidase gene (21) which caused all infected cells to be blue when grown in the presence of X-gal (5-bromo-4-chloro-3-indolyl-P-Dgalactoside). Positive clones were first identified as blue occlusion-negative plaques and then confiied by performing Western slot blots on the cell lysates (data not shown). Three rounds of purification were needed to purify the virus. Production of recombinant Mn peroxidase The recombinant protein was produced in either 2or IO-level cell factories using 0.2 or 1.O L of medium, respectively. Cell factories were seeded to approximately 50% confluency. At 95% confluency, the medium was removed and the cells were infected with the pEVH4 recombinant virus at a multiplicity of infection of 10 plaque forming units per cell. The virus was attached in 100 ml or 500 ml of 1X Grace’s complete, 10% fetal calf serum, and antibiotics for 1 hour and then an equal voiume of the same medium was added. These were incubated at 27°C for 60 hours at which time the medium was removed (concentrated 20 fold, and saved for further analysis) and replaced with fresh serum free and additive free 1X Grace’s with the addition of hemin to a final concentration of 1 ~.&ml. Hemin was dissolved in 50 mM NaOH at a concentration of 1 mg/ml, filter sterilized and added to the medium before addition to the cell factory. These were incubated for an additional 12-14 hours at which time the medium was removed for Mn peroxidase purification. Partial purljkation of recombinant Mn peroxidase The supematant from virus-infected St9 cells was centrifuged (4000 RPM) at 4’C for 10 minutes. The supematant was then concentrated 20 fold (Amicon YMlO membrane) and dialyzed against 5 mM sodium succinate, pH 6.5. This was loaded onto a 100~ml DEAE Biogel A column and eluted with a O-O.5 M NaCl gradient. The active fractions were dialyzed against 5 mM sodium succinate pH 6.5 for storage. Isoelectric focusing Isoelectric focusing (IEF) gels were run using a Pharmacia LKB Multiphor II electrophoresis system. Gels of 5% acrylamide were made by mixing 0.9 ml of pH 4-6 ampholine and 0.6 ml of pH 3.5-5.0 ampholine in a total volume of 30 ml. Mn peroxidase activity was visualized in IEF gels by a modified method of Kersten and Kirk (22). The gel was submerged 200 ml of 82.5 mM sodium succinate, pH 4.5, 82.5 mM sodium lactate, pH 4.5,4.95 mg/ml gelatin, 0,165 mM MnS04 and 0.48 mM phenol red. The color reaction was initiated by the addition of 1 ml of 10 mM H202. After 10 minutes, a second aliquot of H202 was added and the color allowed to develop further. Western blot visualization of the IEF gels was performed by fixing the gels in 0.16 M sulphosalicylic acid, 0.7 M trichloroacetic acid for 30 minutes. The gel was then soaked for 30 minutes in three changes of 12.3 mM Tris HCl, 0.096 M glycine pH 8.6 with 10% methanol and 0.001% SDS at 50°C. The remainder of the procedure was the same as that for an SDS-PAGE Western transfer. Enzyme Activity Assays One unit is defined as one l.trnol of product formed per minute, Mn peroxidase activity was measured by phenol red oxidation except where noted otherwise. The procedure is as described by Glenn and Gold (9) with the exception that a 5.5 ml reaction mix was used and 1 ml was removed every minute and added to 40 pl of 5 M NaOH. Oxidized phenol red was measured at ODelo using an extinction coefficient of 22.0 mM-l . cm-l (23). Vanillylacetone oxidation was assayed by substrate disappearance at 336 nm using an extinction coefficient of 18.6 m~-l. cm-l (24). Guaiacol oxidation was assayed as previously described (25) by measuring tetraguaiacol formation at 465 nm using an extinction coefficient of 26.6 mM-l. cm-l (26). SDS-PAGE gels and Western transfer SDS-PAGE was performed as described by Laemmli (27). Western blots were performed using a polyclonal antibody specific for H4 and the anti-rabbit alkaline phosphatase conjugate. 898
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Results Production of recombinantMnperoxiduse in $9 cells Infection of Sf9 cells with the recombinant&4P-l-containing virus resultedin expressionof active Mn peroxidase. As with the native fungus, the recombinantMn peroxidasewas found in the extracellular medium. At 60 hours post infection, the medium waschangedto 1X Grace’s(serumand additive free) containing hemin at a final concentrationof 1 @ml. Changingthe mediumincreasedenzyme yield and simplified enzyme purification. The mediumwasharvestedat 74 hourspost infection. The activity of the supematant(1 liter) wasmeasuredat 35.7 U/L. Although the addition of hemin elevatedactivity, a low level of activty (lessthan 2 U/L) wasdetectedbefore the mediumchangeand addition of hemin. The enzyme waspurified on a DEAE Biogel-A column for further analyses. Properties of recombinantMnperoxidase The Mr of the recombinantH4, determinedby SDSPAGE, is identical to the native fungal Mn peroxidase. Fig. 1 showsa Western blot of a SDSPAGE gel where the Mn peroxidasewasvisualized using an anti-H4 antibody. The blot showsa singleimmunoreactivebandin the concentratedmediumof infected cells. No immuno-reactive bandswere found in the mediumfrom uninfected Sf9 cells (CC) or vETLP-gal infected cells (VC) (Fig. 1). Purified fungal Mn peroxidaseisozymesH4, H.5, and H3 were run asstandardson the gel.
B P
VC
rMP
nH4
nH5
nH3
rMP
rMP
C
nMP
rMP
nMP
nMP
CC
02
;a Gx
+
+
Fig. 1. SDS-PAGE of recombinant Mn peroxidase (rMP) andnativeMn peroxidase (nMP). Samples weresubjected to SDS-PAGEandvisualizedby Westernblottingtechniques. VC: virus controlcontaining40 pJof supematant from nonrecombinant virus-infectedSf9cells; nH4: 1.8Kgof nativeisozymeH4, rMl? 5 pl of 20-foldconcentrated recombinant supematant; nH5: 1 kg of nativeH5; nH3: 0.8 Fg of nativeH3; andCC: cell controlcontaining40 1.11 of
supematant from non-infectedSt9 cells. Fu Isoelectricfocusinganalyses of recombinant andnativeMn peroxidase.Thecrude recombinant Mn peroxidase(rMP) andthepurifiednativeMn peroxidase (nMP) weresubjected to IEF asdescribedin MaterialsandMethods.Theanode(+) was1M H3PO4 andthecathode(-) was1M NaOH. Samples werevisualizedby Coomassie bluestaining(A), Westernblotting(B), or activity stainingwith phenolred(C). PanelA contained10pl of a 20-foldconcentrated supernatant from recombinant H4 virus-infectedSB cells(rMP) and3.5Kg of purifiednativeH4 from P. chrysosporium(nMP). PanelsB andC contained2 ~1of rMP and0.7 pg of nMP.
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3
Minutes &gJ Time course of phenol red oxidation catalyzed by recombinant Mn peroxidase. Incubations contained 1 ml of crude recombinant supematant in a total of 5.5 ml. Aliquots were removed and assayed for oxidized phenol red as described in Materials and Methods. Incubations contained all of the necessary cofactors (0), minus MnS04 (O), or minus Hz02 (A).
Fig. 2A showsthe total proteinsof the extracellular mediumseparatedby IEF and visualized by Coomassieblue staining. A bandfocusedto approximately the samepI asnative isozyme H4 (~14.6). Western transfer analysisindicated that a bandwith slightly lower p1cross-reactswith an anti-H4-specific antibody (Fig. 2B). The IEF gel, when stainedfor Mn peroxidaseactivity, revealed an intensebandcorrespondingto the immuno-reactiveband(Fig. 2C). There are two minor bandswith phenolred oxidizing activity which are not associatedwith the insect tissue culture. Thesebandsmay be due to partially degradedrecombinantH4 or H4 which has undergonedifferent processing. Substrateprofile of Mn peroxidase Fig. 3 showsthe time coursefor phenol red oxidation by the cruderecombinantMn peroxidasepreparation. Similar to native Mn peroxidase(7), activity is dependenton the presenceof addedMn (II) andHz02 andis linear over time. Phenolred, guaiacol andvanillylacetone, all substratesof the native H4 Mn peroxidasefrom P. chrysosporium (25), are alsooxidized by the recombinantMn peroxidase.Theseactivities are all Mn (II) and Hz& dependentasshown in Table 1.
Discussion We report here the heterologousexpressionof active recombinantMn peroxidasefrom the white-rot fungus P. chrysosporium. The cDNA IMP-l,
previously
identified
Table 1. Activity profile of recombinant Mn peroxidase” Complete minus H202 minus
Mn
(II)
Phenol Red 83 0.8
Guaiacol
Vanillvlacetone
94 0.0
201 1.2
0.0
0.0
5.0
cActivities were determined as per Materials and Methods and am reported in units/L x 1000. The complete reaction mixture contained all the cofactors as described in Materials and Methods. Incubations contained 200 pl of partially purified recombinant Mn peroxidase (rH4).
to encode Mn
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(13), was recombined into the baculovirus
vETLPgal and heterologously expressed in St9 cells. The active recombinant enzyme is secreted into the extracellular medium. The baculovirus expression system has proven to be an excellent technique for expression of eukaryotic enzymes (17,lQ It is especially applicable when various types of post-translational modification are required for enzyme activity (28). This was demonstrated here with the Mn peroxidase. The native H4 undergoes three types of post-translational modification when expressed in P. chrysosporium: i) heme insertion, the Mn peroxidases contain one protoporphyrin IX per enzyme molecule (29); ii) proteolytic processing, the cDNA AMP-1 encodes a leader sequence which directs the enzyme for secretion and is removed (13); and iii) glycosylation, the Mn peroxidases are glycosylated (25). Although we have not purified and characterized the recombinant H4 for the modifications mentioned above, our results suggest that all three are occurring with the baculovirus system. The spectral properties of the recombinant enzyme suggest that heme is inserted. Enzyme activity is associated with a 406 nm-absorbing species (Soret). Furthermore, because heme is part of the active site, it is highly unlikely that activity can be attributed to apoenzyme. Evidence for proper processing of the leader sequence comes from two findings. The first and most direct evidence is that the recombinant enzyme is found in the extracellular medium. The second piece of evidence is that the apparent Mr of the recombinant enzyme, as determined by SDS-PAGE is identical to that of native H4. If the leader sequence was not removed, the Mr would increase approximately 3 kd. Secretion of heterologous proteins using the native leader sequence has been previously observed with the baculovirus expression system and therefore is not a surprising finding here for Mn peroxidase (16,28,30). However, unequivocal demonstation of proper processing of the leader sequences would require purification of the recombinant enzyme and N-terminal amino acid sequencing. The Mn peroxidase isozyme H4 amino acid sequence contains four potential N-linked glycosylation sites (Asn-X-Thr/Ser)
as well as numerous O-linked glycosylation sites (13). Even
though insect cells have been reported to utilize the same N-glycosylation
sites as most vertabrates
and yeast (31), they do not contain sialic acid and practically no galactosyl and sialyl transferases (32). This results in proteins expressed in insect cells having the same core glycosylation, but no complex carbohydrate moieties (31). Little is known about O-linked glycosylation in insect cells (33). The binding of recombinant H4 to a Concanavalin A column suggests that the enzyme is glycosylated (data not shown).
Concanavalin A binds to oligosaccharides containing terminal
mannose residues (34). The only noticeable difference between the recombinant and native enzyme is the p1. The p1 difference would indicate that the processing of the recombinant Mn peroxidase is not identical to that of native Mn peroxidase. The slight differences may be due to a variation in the glycosylation. Nevertheless, the recombinant Mn peroxidase oxidizes the same substrates as the native enzyme (phenol red, guaiacol, and vanillylacetone) All activities are dependent on Mn (II) and Hz% as with the native protein. The relative amount of activity for each substrate is the same for the recombinant and the native Mn peroxidase. Determining whether the recombinant enzyme exhibits similar kinetic parameters of bat and k&& awaits further purification of the enzyme. 901
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Although not essential for production of active enzyme, the addition of hemin to the medium increased activity by at least 15 fold. A similar result was also observed for the expression of mammalian cytochrome P450 in the baculovirus system (18). The addition of hemin to the medium increased the P450 content five to six fold at 72 hours post infection. Addition of hemin to the growth medium at 60 hours post infection correlates with the time of peak recombinant protein production (18). The level of Mn peroxidase activity produced in the baculovirus system is comparable to that produced by the fungus, Although scale up of the baculovims is easier than the fungus, the baculovirus expression system is relatively expensive. Therefore, it will probably not replace the fungus for large-scale enzyme production. However, the baculovirus system will be valuable for mechanistic studies. It will provide purified enzyme free of any possible contamination from other isozymes. There are over ten extracellular lignin-degrading peroxidases produced in P. chysosporim.
Assignment of an activity to a specific isozyme isolated may be problematic due to
possible impurities if the enzyme is isolated from the fungus. The expression of active Mn peroxidase will also permit structure/ function studies using the tools of site-direct mutagenesis.
Acknowledgments This work was supported in part by United States Department of Energy Grant DE-FGOZ 87ER136990 and National Institute of Environmental Health Sciences Grant l-P42ESO4922-01. Ming Tien is a recipient of a Presidential Young Investigator Award from the National Science Foundation (DCB-8657853).
We thank Todd Johnson for his help in transfecting and purifying
the recombinant virus.
References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
Kirk, T. K. and Farrell, R.L. (1987) Ann. Rev. Microbial. 41,465-505. Kirk, T. K., Croan, S., Tien, M., Murtagh, K. E., and Farrell, R. L. (1986) Enzyme Microb. Technol. 8,27-32. Farrell, R. A., Murtagh, K., Tien, M., Mozuch, M. D., and Kirk, T. K. (1989) Enz. Microbial. Tech. 11, 322-328. Tien, M. and Kirk, T. K. (1983) Science 221, 661-663. Glenn, J. K., Morgan, M. A., Mayfield, M. B., Kuwahara, M., and Gold, M. H. (1983) Biochem. Biophys. Res. Commun. 114, 1077-1083. Paszczynski, A., Huynh, V.-B., and Crawford, R. (1985) FEMS Microbial. Lett., 28, 119128. Kuwahara, M., Glenn, J. K., Morgan, M. A., Gold, M. H. (1984), FEBS Lett., 169, 247249. Kirk, T. K., Tien, M., Kersten, P. J., Mozuch, M. D., and Kalyanaraman, B. (1986) Biochem. J. 236,279-287. Glenn, J. K. and Gold, M. H. (1985) Arch. Biochem. Biophys. 242, 329-341. Tien, M. and Tu, C.-P. D. (1987) Nature 326,520-523; also Nature 328,742. de Boer, H. A., Zhang, Y. Z., Collins, C., and Reddy, C. A. (1987) Gene 60,93-102. Andrawis, A., Pease, E. A., Kuan, I., Holzbaur, E., and Tien, M. (1989) B&hem. Biophys. Res. Comm. 162, 673-680. Pease, E. A., Andrawis, A., and Tien, M. (1989) J. Biol. Chem. 264, 13531-13535. Pribnow, D., Mayfield, M. B., Nipper, V. J., Brown, J. A., Gold, M. (1989), J. Biol. Chem. 264,5036-5040. Andrawis, A., Pease, E. and Tien, M. (1990) in Biotechnology in Pulp and Paper Manufacture Applications and Fundamental Investigations (T. K. Kirk and H-m. Chang, ed.) Butterworth-Heinemann., 601-613, Butteworth-Heinemann, Boston, 902
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