Large-scale purification of rat brain nitric oxide synthase from baculovirus overexpression system

420

NO SYNqT-IASE

141]

well as the biological control mechanisms involved may translate into a better understanding of the pathophysiology and, ultimately, therapy of disorders associated with an enhanced formation of NO due to expression of iNOS.

[41] Large-Scale Purification of Rat Brain Nitric Oxide Synthase from Baculovirus Overexpression System By BERND MAYER, PETER KLATT, BARBARA M . LIST, CHRISTIAN HARTENECK, a n d KURT SCHMIDT

Introduction Oxidation of L-arginine to L-citrulline and nitric oxide (NO) is catalyzed by at least three different NO synthase (NOS; EC 1.14.13.39) isozymes: the constitutively expressed neuronal and endothelial isoforms, which require Ca 2+ calmodulin for activity, and a Ca2+-independent form, which is expressed in most mammalian cells in response to cytokines and endotoxin) -3 Albeit regulated in a different manner, the NOS isoforms identified so far are biochemically similar. They exist as homodimers under native conditions, with subunit molecular masses ranging from 130 kDa (endothelial and inducible NOSs) to 160 kDa (neuronal NOS). L-Arginine oxidation and reductive activation of molecular oxygen is catalyzed by a cysteine thiol-ligated P450-1ike prosthetic heme group localized in the N-terminal half of the protein. 4,5 This part of the enzyme is thus referred to as the oxygenase domain of NOS. The C-terminal half of the enzyme, separated from the oxygenase domain by a binding site for calmodulin, contains the flavins FAD and FMN that serve to shuttle reducing equivalents from the cofactor NADPH to the heme. This reductase domain of NOS shows pronounced sequence similarities to cytochrome P450 reductase 6 and exhibits similar catalytic activities.7 These results suggest that NOSs represent 1 M. A. Marietta, J. Biol. Chem. 268, 12231 (1993). 2 R. G. Knowles and S. Moncada, Biochem. J. 298, 249 (1994). 3 B. Mayer, Cell Biochem. Funcr 12, 167 (1994). 4 p. F. Chen, A. L. Tsai, and K. K. Wu, J. Biol. Chem. 269, 25062 (1994). 5 M. K. Richards and M. A. Marietta, Biochemistry 33, 14723 (1994). 6 D. S. Bredt, P. M. Hwang, C. E. Glatt, C. Lowenstein, R. R. Reed, and S. H. Snyder, Nature (London) 351, 714 (1991). 7 p. Klatt, B. Heinzel, M. John, M. Kastner, E. B6hme, and B. Mayer, J. Biol. Chem. 2679 11374 (1992).

METHODS IN ENZYMOLOGY,VOL.268

Copyright© 1996by AcademicPress,Inc. AUrightsof reproductionin any formreserved.

[41]

PURIFICATION OF RECOMBINANTBRAINNO SYNTHASE

421

self-sufficient cytochrome P450s with both reductase and oxygenase activities associated with one single protein. In contrast to the classic microsomal cytochrome P450 systems, however, NOSs require the pteridine tetrahydrobiopterin as an additional cofactor with as yet unknown function, s-l° We have worked out a method for purification of rat brain NOS overexpressed in a baculovirus/insect cell system, u Here we describe an upscaled version of this protocol yielding about 100 mg of the pure enzyme from 3000-ml Sf9 (Spodoptera frugiperda fall armyworm ovary) cell cultures. Plasmid Construction The cDNA encoding for rat brain NOS (Genbank accession No. X59949), 6 a generous gift from Drs. D. S. Bredt and S. H. Snyder, was cloned in the baculovirus expression vector pVL 1393. Cloning was performed according to standard laboratory protocols and is only briefly described here. 12To reduce the 5' noncoding region of the original cDNA, a Sse8387I fragment of NOS (bp 308-4241) was cloned in the PstI site of pBluescript S K ( - ) (Stratagene, La Jolla, CA). A clone with the C-terminal part next to the NotI site of the multiple cloning site of pBluescript was selected and cut with EheI (position 1560) and NotI (multiple cloning site of pBluescript). The 3139-bp EheI (position 1560) and NotI (position 4699) fragment of the original cDNA encoding the C-terminal part was then ligated in the cloning intermediate. The resulting cDNA was cloned in the transfer vector pVL 1393 using EcoRI/NotI. Infection of Sf9 Cells with Recombinant Baculovirus Fall armyworm ovary cells (Spodoptera frugiperda; Sf9) are obtained from the ATCC (Rockville, MD; CRL 1711). Cells are grown in TC-100 medium (Sigma, St. Louis, MO) supplemented with 10% fetal calf serum (SEBAK GmbH, Suben, Austria), amphotericin B (1.25 mg/liter), penicillin (100,000 U/liter), streptomycin (100 mg/liter), and 0.2% Pluronic F-68 (Sigma) under continuous shaking (60-70 rpm) using an orbital shaker. Optimal growth (doubling time of ~24 hr) is achieved at cell densities ranging from 5 × 105 to 2 × 10 6 cells/ml and a temperature of 27° + 1°. s p. Klatt, M. Schmid, E. Leopold, K. Schmidt, E. R. Werner, and B. Mayer, J. Biol. Chem. 269, 13861 (1994). 9 B. Mayer, P. Klatt, E. R. Werner, and K. Schmidt, J. Biol. Chem. 270, 655 (1995). 10B. Mayer and E. R. Werner, Naunyn-Schmiedeberg's Arch. Pharmacol. 351, 453 (1995). u C. Harteneck, P. Klan, K. Schmidt, and B. Mayer, Biochem. J. 304, 683 (1994). 12j. Sambrook, E. F. Fritsch, and T. Maniatis, "Molecular Cloning: A Laboratory Manual." Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989.

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NO SVNTrtASE

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The recombinant virus is generated by cotransfection of Sf9 cells with the expression vector (see section on plasmid construction) and BaculoGOLD baculovirus DNA (Dianova, Hamburg, Germany) using the lipofection method. 13 Positive viral clones are isolated by plaque assay and identified by the ability to direct the expression of NOS as revealed by appearance of a 150- to 160-kDa band in immunoblots of the cell extracts using a polyclonal antibody against brain NOS (ALEXIS, Lfiufelfingen, Switzerland) for detection. The purified virus is then amplified and the titer estimated by plaque assay. For expression of NOS, the culture medium is supplemented with 20% (w/v) fetal calf serum, and Sf9 cells (4.5 × 109 cells/3000 ml) are infected with the recombinant baculovirus at a ratio of 5 pfu (plaqueforming units) per cell in the presence of 4 mg/liters heroin (Sigma). Stock solutions (2 mg/ml) of hemin are prepared in 0.4 M NaOH/ethanol (1 : 1, v/v). After 48 hr, cells are harvested by centrifugation for 3 rain at 1000 g and washed with 500 ml of serum-free TC-100 medium. Finally, cells are resuspended in buffer A (see section on solutions and buffers) to give a final volume of 100 ml and used for enzyme preparation as described below. Comments

(1) For preparation of NOS as described in this chapter, we have used three 1800-ml Fernbach flasks, each containing not more than 1000 ml of cell suspension to ensure sufficient oxygenation of the cells. (2) In initial experiments we found that culture of Sf9 cells at ambient temperature not only slowed down cell growth but also markedly reduced NOS expression levels. It is strongly recommended, therefore, to use an incubator with a built-in cooling system to keep the cells at 27°. (3) Addition of hemin during infection turned out to be crucial to obtain functionally intact NOS. Interestingly, we observed even higher expression levels in cells infected in the absence of hemin; however, a large part (>90%) of the enzyme remained insoluble under these conditions, and the remaining soluble part was only poorly active, apparently due to low heme content (-0.2 tool per mole 160-kDa subunit). (4) As previously observed by others, 14'15we found that hemin cytotoxicity was prevented by infecting cells in the presence of 20% (w/v) fetal calf serum. (5) Supplementation of the culture medium with either riboflavin (0.1 mM) or FAD and FMN (0.1 mM each) increased the amount of flavins bound to the purified enzyme from 0.2 to 0.7-0.9 mol per mole 160-kDa subunit. This is not essential, however, because 13D. R. Groebe, A. E. Chung, and C. Ho, Nucleic Acids Res. 18, 4033 (1990). 14 A. Asseffa, S. J. Smith, K. Nagata, J. Gillette, H. V. Gelboin, and F. J. Gonzalez, Arch. Biochem. Biophys. 274, 481 (1989). 1~F. J. Gonzalez, S. Kimura, S. Tamura, and H. V. Gelboin, Methods Enzymol. 206, 93 (1991).

[41]

PURIFICATION OF RECOMBINANT BRAIN

NO

SYNTHASE

423

flavin-deficient NOS is readily reconstituted by addition of FAD and FMN during incubation, u (6) It should be considered that specific NOS activities in crude preparations of Sf9 cells decrease at increasing concentrations of total protein. (7) Occasionally and without any obvious reason, infection of Sf9 cells did not yield satisfactory NOS expression levels. It is recommended, therefore, to determine NOS activity in homogenates or supernatants prepared from a small aliquot of the infected cells prior to enzyme purification.

Enzyme Purification The protocol for purification of rat brain NOS involves two sequential affinity chromatography steps. First the enzyme is bound to 2',5'-ADPSepharose 4B (Pharmacia, Piscataway, NJ) and eluted with excess NADPH or 2'-AMP (see comments below). Subsequently it is chromatographed in the presence of Ca 2+ over calmodulin-Sepharose 4B (Pharmacia) and eluted by chelating Ca t+ with excess EGTA. The following buffers and solutions should be prepared 1 day before enzyme preparation in doubledistilled water and be adjusted to pH 7.4 at ambient temperature (20°-25°). The buffers are passed through a 0.22-/xm filter and kept overnight at 4°-6 °.

Solutions and Buffers 2-Mercaptoethanol should be added to buffers A - E immediately before use. 1.5 ml of 0.5 M EDTA (298 mg NaaEDTA. HzO) 10 ml of 200 mM CaCI2 (296 mg CaCI2.2H20) Buffer A (500 ml): 20 mM triethanolamine hydrochloride (TEA; 1.86 g), adjusted to pH 7.4 with 5 M NaOH; 0.5 mM EDTA (0.5 ml EDTA stock); 12 mM 2-mercaptoethanol (0.5 ml) Buffer B (250 ml): 50 mM TEA (2.32 g) and 0.5 M NaC1 (7.31 g) adjusted to pH 7.4 with 5 M NaOH; 0.5 mM EDTA (0.25 ml EDTA stock); 12 mM 2-mercaptoethanol (0.25 ml) Buffer C (250 ml): 50 mM TEA (2.32 g) and 150 mM NaC1 (2.19 g) adjusted to pH 7.4 with 5 M NaOH; 0.5 mM EDTA (0.25 ml EDTA stock); 12 mM 2-mercaptoethanol (0.25 ml) Buffer D (500 ml): 20 mM Tris-HC1 (1.21 g Tris base) and 150 mM NaCI (4.38 g) adjusted to pH 7.4 with 6 M HC1; 2 mM CaC12 (5 ml CaCl2 stock); 12 mM 2-mercaptoethanol (0.5 ml) Buffer E (250 ml): 20 mM Tris-HC1 (0.61 g Tris base), 150 mM NaC1 (2.19 g), and 4 mM EGTA (0.38 g) adjusted to pH 7.4 with 6 M HC1; 12 mM 2-mercaptoethanol (0.25 ml)

424

NO SYNTHASE

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Column Chromatography Geometry of the columns is probably not critical, but bed volumes should be adapted to the scale of the enzyme preparations to avoid dilution of the protein or overloading of the columns. We have used two 20 × 2.5 cm Econo-Columns (Bio-Rad, Richmond, CA) equipped with flow adaptors and filled with the affinity resins to give bed volumes of 35 ml each. Flow rates are 2 ml/min throughout enzyme preparation. Columns are equilibrated with 4 bed volumes (140 ml) of buffer A (2',5'-ADP-Sepharose) or buffer D (calmodulin-Sepharose) prior to enzyme purification. Regeneration of the columns is performed at flow rates of 1 ml/min. The 2',5'-ADP-Sepharose is washed with 100 ml of alkaline buffer (0.1 M Tris, 0.5 M NaC1, 0.1% (w/v) NAN3, pH 8.5) followed by 100 ml of acidic buffer (0.1 M sodium acetate, 0.5 M NaCI, 0.1% (w/v) NAN3, pH 4.5). This cycle is repeated three times, and finally the column is washed with 100 ml of double-distilled water containing 0.1% (w/v) NaN3 for storage. Calmodulin-Sepharose is regenerated with 200 ml of 50 mM Tris, 2 mM EGTA, 1 M NaC1, 0.1% NAN3, pH 7.5, and finally equilibrated with 100 ml of 50 mM Tris, 2 mM CaCI2, 150 mM NaC1, 0.1% NAN3, pH 7.5, for storage.

Purification Procedure Infected cells suspended in buffer A and kept on ice are sonicated four times for 10 sec each time at 150 W, homogenates are centrifuged for 15 min at 30,000 g (Sorvall SS-34 rotor; 17,000 rpm), and the supernatant is collected. To increase recovery of NOS by about 50%, the pellet is washed once with buffer A (final volume 50 ml). The combined supernatant is loaded onto the equilibrated 2',5'-ADP-Sepharose column, which is subsequently washed with 70 ml of buffer B followed by 35 ml of buffer C. Elution of bound enzyme is performed with 90 ml of 10 mM N A D P H in buffer C, and fractions (3 ml) containing NOS (visible as red-brown color) are pooled to give about 60 ml of 2',5'-ADP-Sepharose eluate. The 2',5'-ADP-Sepharose eluate is adjusted to 2 mM CaCI2 by addition of CaClz stock solution (1%, v/v) and passed over the calmodulin-agarose column. The column is washed with 105 ml of buffer D, and the enzyme is eluted with 105 ml of buffer E. Fractions (3 ml) containing NOS (visible as red-brown color) are pooled to give about 30 ml of eluate, containing approximately 4 mg of purified NOS per milliliter. If required, the final eluate can be further concentrated up to 20 mg/ml using Centricon-30 microconcentrators (Amicon, Danvers, MA). The enzyme should be stored at - 7 0 °.

[41]

PURIFICATION OF RECOMBINANT BRAIN

NO

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Comments

(1) The method described is rather insensitive to downscaling, albeit the relative amounts of recovered enzyme may be lower at smaller scales. Initially, we have purified the enzyme from 400-ml cultures of infected cells and obtained results similar to those described here, u and sometimes we perform minipreparations from 150-ml cultures yielding approximately 1 mg of pure NOS. If required, several of these minipreparations can be done simultaneously, using small disposable plastic columns with a bed volume of 0.5 ml for affinity chromatography. (2) Yasukochi and Masters 16 have described elution of cytochrome P450 reductase from 2',5'-ADPSepharose with 2'-AMP, which is considerably less expensive than NADPH. In accordance with this previous report, we found that recombinant rat brain NOS was efficiently eluted from the affinity resin in the presence of 20 mM 2'-AMP (Sigma, containing -50% of inactive 3'-AMP) and 0.5 M NaC1 without interference with subsequent binding of the enzyme to calmodulin-Sepharose. (3) Purified NOS is sensitive to repeated freezing and thawing. Thus, the final eluate should be frozen in small aliquots at reasonable protein concentrations. (4) The enzyme is unstable if diluted or assayed in the absence of either serum albumin (2-10 mg/ml) or certain detergents. Routinely we dilute the enzyme in the presence of l mM 3-[(3-cholamidopropyl)dimethylammonio]-l-propanesulfonate (CHAPS), giving final concentrations of 0.2 mM CHAPS in enzyme assays. Results of Representative Purification We now describe the results of a typical purification of NOS from 3000ml cultures of Sf9 cells. Protein is determined with the method of Bradford 17 using bovine serum albumin (BSA) as standard. Unless otherwise indicated, NOS activity is assayed as conversion of [3H]arginine to [3H]citrulline as described in detail by Mayer et aL 18 Figure 1 shows a Coomassie blue-stained sodium dodecyl sulfate (SDS)polyacrylamide gel of Sf9 cell supernatant (lane B), 2',5'-ADP-Sepharose eluate (lane C), and the final calmodulin-Sepharose eluate (lane D). Nitric oxide synthase appears as a 155-kDa band on the gels, which is in good accordance with the molecular mass calculated from the amino acid sequence (160 kDa). 6 Densitometric analysis of the gel indicated that NOS accounted for approximately 6% of total soluble cell protein. Data on 16 y . Yasukochi and B. S. S. Masters, J. Biol. Chem. 251, 5337 (1976). 17 M. M. Bradford, Anal, Biochem. 72, 248 (1976). 18 B. Mayer, P. Klatt, E. R. Werner, and K. Schmidt, Neuropharmacology

33, 1253 (1994).

426

N O SYNTHASE kDa

A

200-

B

C

D

-"" m

94

[4 11

t

m

67m 43

m

30m

Front - -

I

~

t

~t

FIG. 1. Coomassie blue-stained SDS-polyacrylamide gel (10%) of marker proteins (lane A), supernatant of rat brain NOS-recombinant baculovirus-infected Sf9 cells (lane B), 2',5'ADP-Sepharose eluate (lane C), and calmodulin-Sepharose eluate (lane D). Occasionally, the 2',5'-ADP-Sepharose eluates contained some additional bands in the range of 40-90 kDa, accounting for 2-5% of total stained protein. These minor bands were never observed, however, in the calmodulin-Sepharose eluates.

e n z y m e activities a n d p r o t e i n r e c o v e r y d u r i n g p u r i f i c a t i o n a r e s u m m a r i z e d in T a b l e I. A b o u t 3 g of s o l u b l e p r o t e i n was o b t a i n e d f r o m 4.5 × 109 ceils, a n d N O S a c t i v i t y was 0 . 0 5 1 / x m o l c i t r u l l i n e / m g / m i n in t h e cell s u p e r n a t a n t . C h r o m a t o g r a p h y o v e r 2 ' , 5 ' - A D P - S e p h a r o s e y i e l d e d a b o u t 150 m g o f p r o t e i n w i t h a specific a c t i v i t y of 0 . 9 / z m o l c i t r u l l i n e / m g / m i n , w h i c h is s i m i l a r to t h e activities p r e v i o u s l y d e s c r i b e d for N O S p u r i f i e d f r o m r a t a n d p o r c i n e

TABLE

I

PURIFICATION OF RECOMBINANT RAT BRAIN NITRIC OXIDE SYNTHASE a

Fraction 30,000 g supernatant 2',5'-ADP-Sepharose Calmodulin-Sepharose Centricon concentrate

Total Specific Volume Protein activity activity Purification Yield (ml) (mg) (/zmol/min) (/xmol/mg/min) (-fold) (%) 85 60 30 4.6

3080 156 123 83

157 141 117 91

0.051 0.90 0.95 b 1.10b

1 18 19 21

100 90 75 58

"Nitric oxide synthase was purified from 4.5 × 109 Sf9 cells that had been infected with rat brain NOS-recombinant baculovirus for 48 hr in 3000 ml culture medium supplemented with 4 mg/liter heroin. Enzyme activity was measured at 37° for 10 rain as conversion of [3H]arginine (0.1 mM) to [3H]citrulline. Results from a representative purification are shown. b Differences in specific activities of NOS prior and after protein concentration are not statistically significant when different purifications are compared (n = 3; p > 0.20).

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brain. 19,2°Further purification over calmodulin-Sepharose resulted in about 2-fold concentration of the protein and sometimes slightly enhanced its specific activity. Loss of enzyme was about 20% in this step, giving an overall yield of 75% of NOS activity over crude supernatants. In the calmodulin-Sepharose eluate, the NOS concentration was 4 mg/ml corresponding to a 25/~M solution of the subunits. For biophysical characterization, the enzyme was further concentrated by centrifugation in Centricon-30 microconcentrator devices. Typically, concentration of NOS to 15-20 mg/ml results in 20-30% loss of enzyme without significant change in specific activity. Purified NOS exhibited a Km for L-arginine of 5/~M and a Vmax of 1.2--1.5 tzmo1 L-citrulline/mg/min, indicating that recombinant rat brain NOS is kinetically similar to the enzyme purified from porcine brain. 21 Determination of bound prosthetic groups 22 showed almost stoichiometric presence of heme (molar ratio 0.90-0.95 per subunit), but the flavins and tetrahydrobiopterin were present at substoichiometrical amounts (0.2 and 0.4 tool per mole of subunit, respectively), u As indicated in the comments for cell infection (see above), the flavin content of purified NOS can be increased to a molar ratio of 0.7-0.9 per subunit when cells are infected in the presence of 0.1 m M riboflavin or F A D / F M N , whereas the amount of bound tetrahydrobiopterin is not increased by infection of cells in the presence of various pteridines or sepiapterin, a precursor of tetrahydrobiopterin biosynthesis. J9D. S. Bredt and S. H. Snyder, Proc. Natl. Acad. Sci. U.S.A. 87, 682 (1990). 2oB. Mayer, M. John, and E. B6hme, FEBS Letr 277, 215 (1990). 2J p. Klatt, K. Schmidt, G. Uray, and B. Mayer, J. Biol. Chem. 268, 14781 (1993). 2-~p. Klatt, K. Schmidt, E. R. Werner, and B. Mayer, Methods Enzymol. 268, Chap. 35, 1996 (this volume).

[42] C l o n e d

and Expressed Nitric Oxide Synthase Proteins

B y H O U H U I X I A a n d D A V I D S. B R E D T

Cloning of Nitric Oxide S y n t h a s e Genes Initial biochemical characterization of nitric oxide synthase (NOS; E C 1.14.13.39) activities in mammalian tissues suggested the existence of at METHODS IN ENZYMOLOGY, VOL. 268

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