[36]
PURIFICATIONOF MGSA
373
[36] P u r i f i c a t i o n a n d C h a r a c t e r i z a t i o n o f R e c o m b i n a n t Melanoma Growth Stimulating Activity B y H . GREG THOMAS, JIN H E E H A N , E D D Y BALENTIEN, RIK DERYNCK, RODOLFO BORDONI, a n d A N N RICHMOND
Introduction
Melanoma growth stimulatory activity (MGSA) was originally purified from extracts of serum-free culture medium of Hs294T human malignant melanoma cells and a variety of melanoma tumors. MGSA exerts an autocrine-like growth effect for melanoma cells, j Only recently was a method described that provided adequate amounts of purified protein for complete characterization of MGSA secreted by the Hs294T melanoma cells. 2 This factor is structurally related to various polypeptides in the/3thromboglobulin superfamily which are 8-10 kDa basic heparin-binding proteins. 2 Subsequent isolation and characterization of cDNAs revealed a predicted length of 73 amino acids proteolytically derived from a 107 residue precursor. In addition, the nucleotide sequence of the MGSA gene possesses similarity with the human "gro" gene and the PDGF-inducible gene K C . 3-5 Northern hybridization demonstrates that MGSA/gro gene expression is not restricted to melanoma cells but is found in a variety of normal and transformed cells of different origin. 3 The recently optimized procedure for the purification of MGSA from Hs294T cell culture medium required multiple processes which were laborious and time consuming. Although adequate amounts of protein were obtained for complete characterization and initial biological studies, the total yield of homogeneous material was low for the time and expense required. Therefore, a mammalian MGSA expression system has been developed in order to produce larger quantities of MGSA. The expression system utilizes a plasmid vector construct which places the MGSA gene under the control of the cytomegalovirus promoter in Chinese hamster ovarian (CHO) cells. I A. Richmond and H. G. Thomas, J. Cell. Physiol. 129, 375 (1986). 2 H. G, Thomas and A. Richmond, Mol. Cell. Endocrinol. 57, 69 (1988). 3 A. Richmond, E. Balentien, H. G. Thomas, G. Flaggs, D. E. Barton, J. Spiess, R. Bordoni, U. Francke, and R. Derynck, EMBO J. 7, 2025 (1988). 4 A. Anisowicz, L. Bardwell, and R. Sager, Proc. Natl. Acad. Sci. U.S.A. 84, 7188 (1987). 5 p. Oquendo, J. Alberta, D. Wen, J. L. Graycar, R. Derynck, and C. D. Stiles, J. Biol. Chem. 264, 4133 (1989).
METHODS IN ENZYMOLOGY, VOL. 198
Copyright 63 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
374
OTHER GROWTH FACTORS AND GROWTH INHIBITORS
[36]
We compare here the purification of MGSA to homogeneity from the Hs294T cells and from stable CHO transfectants which secrete high levels of human MGSA. The proteins obtained from these sources are compared as to size, biological activity, N-terminal amino acids, and recognition by antibodies raised against proteins derived from both cell sources, demonstrating the identical properties and activities of the proteins. Sources of Growth Factors Tissue culture materials and media are supplied as previously described. I Hs294T cells are obtained from the ATCC (Rockville, MD), and a subline is selected which grows well on serum-free Ham's F10 medium without growth factor supplements. Transfected Chinese hamster ovary cells are selected in complete medium lacking glycine, hypoxanthine, and thymidine and supplemented with 10% dialyzed fetal bovine serum as described. 6 Assay Methods for Characterizing Melanoma Growth Stimulatory Activity
[3H]ThymidineIncorporation. MGSA bioassays are performed by measuring stimulation of [3H]thymidine incorporation in serum-depleted low density cultures of Hs294T human malignant melanoma cells according to a modification of the procedures of Iio and Sirbasku 7 as previously described. 1 Cells (8000) are plated into 28 × 61 mm glass scintillation vials (Wheaton, Millville, NJ, #22528) in 2 ml of Ham's F10 medium supplemented with 10% fetal bovine serum. Twenty-four hours later, cells are washed with 2 ml of phosphate-buffered saline (PBS: 8 g NaC1, 1.15 g Na2HPO 4, 0.2 g KCI, 0.2 g KH2PO 4, 0.1 g MgCI 2 • 6H20, 0.1 g CaCI z) and placed on serum-free F10 medium containing HEPES (30 mM) and ovalbumin (I0/zg/ml). After a 24-hr incubation in serum-free medium the medium is aspirated, and aliquots of growth factor are added in a binding buffer composed of 2 ml of serum-flee FI0 with HEPES (30 mM) and ovalbumin (10/zg/ml). Eight hours later [3H]thymidine (5/xCi) is added to each vial, and the incubation is continued for an additional 16 hr. The reaction is stopped by the addition of methanol/ethanol (3 : 1). Unincorporated [3H]thymidine is removed by repeated washing with methanol, then 10 ml of Scintiverse II is added to each vial, and the radioactivity incorpo6 R. Derynck, E. Balentien, J. H. Han, H. G. Thomas, D. Wen, A. K. Samantha, C. O. Zachariae, P. R. Griffin, R. Brachmann, W. L. Wong, K. Matsushima, and A. Richmond, Biochemistry 29, 10225 (1990). 7 M. Iio and D. Sirbasku, Cold Spring Harbor Conf. Cell Proliferation 9, 751 (1982).
[36]
PURIFICATIONOF MGSA
375
rated into DNA is counted in a Beckman liquid scintillation counter. Results are compared to controls that receive only binding buffer. Cell Number Assay. MGSA bioactivity can also be assessed in a cell number assay, j Hs294T cells (8000) are seeded into glass scintillation vials as described for the [3H]thymidine assay. After 72 hr the medium is aspirated, and the cells are washed twice with PBS and placed on serumfree F10 containing HEPES (30 raM) and ovalbumin (I0/zg/ml). Fortyeight hours later the medium is aspirated, and dilutions of MGSA in a binding buffer composed of serum-free F10 with HEPES (30 mM) and ovalbumin (10 ~g/ml) are added. On the third and sixth day after growth factor additions, cells are released by brief treatment with trypsin, and the cell number is determined from an aliquot of suspended cells counted on a hemocytometer. Denaturing Electrophoresis. Sodium dodecyl sulfate (SDS) gradient gels (linear, 12-18% polyacrylamide) are prepared as described by Laemmli 8 and poured into slabs 0.75 mm thick. Samples of MGSA from various steps of the purification are brought to less than 10/xl or dryness in a Speed Vac (Savant, Hicksville, NY) or a lyophilyzer. Twice-concentrated SDS sample buffer ( - 2 0 txl) is added, and the samples are electrophoresed at 35 mA constant current. After electrophoresis the gels are fixed and stained with Coomassie blue R-250 and/or silver (Bio-Rad, Richmond, CA, silver stain kit).
Purification of Natural Melanoma Growth Stimulatory Activity
Step l: Extraction from Hs294T Conditioned Medium. Natural MGSA was initially purified from serum-flee culture medium conditioned by confluent cultures of the Hs294T human melanoma cell line as previously described. 1 The serum-free conditioned medium is collected from P-150 culture flasks using sterile technique, and the protease inhibitor Trasylol (aprotinin) (Sigma, St. Louis, MO) is added (117 kallikrein inhibitory units per liter). The medium is centrifuged at 15,000 g to remove floating cells or cellular debris, then the supernatant is ultracentrifuged (100,000 g) for 30 min to remove subcellular organelles. The resulting supernatant is lyophilized. When 20 liters of conditioned medium has been processed in this manner, the lyophilized powder is extracted with 1 N acetic acid at room temperature, dialyzed exhaustively against 0.17 N acetic acid at 4 °, lyophilized, and stored at - 8 0 °. Step 2: BioGel P-30 Chromatography. Acetic acid extracts of approximately 20 liters of lyophilized Hs294T conditioned medium are dissolved 8 U. K. Laemmli, Nature (London) 227, 680 (1972).
376
[36]
OTHER GROWTH FACTORS AND GROWTH INHIBITORS
2.5
"~
2.0
i
1.5
H
= 1.o ~
,6O
~J~
0.5..]
2O
20
40
60
Fraction Number
FIG. I. Reversed-phase HPLC of MGSA on a p~Bondapak CI8 column. Fractions eluting from the BioGei P-30 column between the 6K and 14K standards were combined, lyophilized, and dissolved in 2 ml o f 6 : 94 : 0.05 acetonitrile/water/trifluoroacetic acid. The sample was applied to a p,Bondapak CI8 column equilibrated with the same buffer. The gradient consisted o f 6-60% acetonitrile for 60 min at 25 ° with a flow rate of 1.5 ml/min. Sixty fractions were collected. Aliquots o f the fractions were used for MGSA bioassays.
in 30 ml of 1 N acetic acid and clarified by ultracentrifugation (100,000 g for 30 min). The resulting supernatant, containing approximately 200 mg of protein, is applied to a 2.5 x 90 cm BioGel P-30 column previously equilibrated with 1 N acetic acid and calibrated with 25K, 14K, and 6K polypeptides. The protein is eluted with 1 N acetic acid at a flow rate of approximately 9 ml/hr at room temperature. Absorbance is monitored at 280 nm, and 80-drop fractions are collected. Greater than 95% of the protein initially loaded onto the column elutes prior to the 25K marker. MGSA bioactivity elutes broadly and resides in two major molecular weight regions (14,000-25,000 and 5000-10,000). 9 The apparent high molecular weight (14,000-25,000) MGSA fractions are combined, and the apparent low molecular weight (5000-10,000) MGSA fractions are combined, with the pools being labeled A and B, respectively. Step 3: Initial Reversed-Phase Chromatography. Pools A and B from the BioGel P-30 column are lyophilized separately and dissolved in 1-2 ml of acetonitrile/water/trifluoroacetic acid (6 : 94 : 0.05). Each sample is injected into a/zBondapak Cj8 cartridge column (8 x 100 mm) in a Z module and eluted with a 60-min linear gradient of acetonitrile/water/ trifluoroacetic acid (6 : 94 : 0.05 to 60 : 40 : 0.05 (v/v), pH 2.2) at a flow rate of 1.5 ml/min (Fig. 1). Absorbance is monitored at 206 nm, and 1.5-ml fractions are collected. 9 A. Richmond, H. G. Thomas, and R. G. B. Roy, this series, Vol. 146, p. i 12.
[36]
PURIFICATIONOF MGSA
377
Step 4: Heparin-Sepharose Chromatography. Heparin-Sepharose chromatography has recently been utilized as an alternative to preparative SDS-PAGE for the subsequent purification of MGSA from the initial reversed-phase HPLC fractionation. 2 Bioactive fractions eluting between 31 and 38% acetonitrile are pooled, lyophilized, redissoived in 10 mM Tris, pH 7.5, and applied to a 10 x 50 mm column of heparin-Sepharose CL6B equilibrated in the same buffer. Material that does not bind to heparinSepharose is removed by continuous flow of 10 mM Tris, pH 7.5. A 200ml linear gradient of 10 mM Tris, pH 7.5, to 10 mM Tris, 1 M sodium chloride, pH 7.5, is followed by elution with 100 ml of 10 mM Tris, 2 M sodium chloride, pH 7.5, and finally elution with 100 ml of 6 M guanidine hydrochloride. Absorbance is monitored at 280 nm, at a sensitivity of 0.1 absorbance units full scale (AUFS). The flow rate is 0.5 ml/min, and 8-ml fractions are collected. Fractions are pooled based on the absorbance at 280 nm, and lyophilized aliquots are subjected to bioassay, The material eluting from 0.1 to 0.3 M sodium chloride provides the more significant stimulation in the cell number assay (-200% control) and is utilized for subsequent reversed-phase HPLC purification. Step 5: Additional Reversed-Phase Chromatography. Bioactive material from the heparin-Sepharose column is subjected to reversed-phase HPLC fractionation on a Vydac C~8 column using a linear gradient of 6 : 94 : 0.05 to 60 : 40 : 0.05 (v/v) acetonitrile/water/trifluoroacetic acid. Absorbance is monitored at 214 nm, and 2-ml fractions are collected. Aliquots of the 2-ml fractions are analyzed by SDS-PAGE and/or bioassay prior to further chromatography. The fractions are then rechromatographed on the Vydac column using a mobile phase containing trifluoroacetic acid or heptafluorobutyric acid. The linear gradients employed consist of 25 : 75 : 0.05 to 35 : 65 : 0.05 acetonitrile/water/trifluoroacetic acid or 25 : 75 : 0.05 to 45 : 55 : 0.05 acetonitrile/water/heptafluorobutyric acid. Complete resolution of the individual moieties requires an additional two or three HPLC fractionations using the above gradient containing trifluoroacetic acid but only one additional run when using heptafluorobutyric acid. These modifications significantly increase the yield of homogeneous MGSA from less than 1/~g using preparative SDS-PAGE to 5-10/zg when using these modifications. Purification of Recombinant Melanoma Growth Stimulating Activity The purification of recombinant MGSA (rMGSA) from the CHO clone 7 cells utilizes many of the procedures previously mentioned.
Step 1: Extraction from CHO Clone 7 Conditioned Medium. Five liters
378
O T H E R G R O W T H FACTORS A N D G R O W T H I N H I B I T O R S
[36]
of serum-free conditioned medium from the CHO clone 7 cells transfected with the expression plasmid pCMV-M236 is concentrated by lyophilization, prior to dialysis against 0.17 N acetic acid. Step 2: BioGel P-30 Chromatography. The concentrate from Step 1 is then subjected to BioGel P-30 chromatography as previously described. Step 3: Heparin-Sepharose Chromatography. Fractions eluting from the BioGel P-30 column between the ribonuclease A and insulin markers (13.7K and 6K, respectively) are pooled, lyophilized, and subjected to heparin-Sepharose affinity chromatography as previously described. 2 Step 4: Reversed-Phase Chromatography. Fractions eluting from the heparin-Sepharose column with 0.5 M NaCI are pooled, lyophilized, and dissolved in a solution containing acetonitrile, water, and trifluoroacetic acid (6:94:0.05) (v/v). Subsequent reversed-phase HPLC is performed on a Vydac Hi-Pore C~8 column using a 60-min linear gradient from 6 to 60% acetonitrile. 2 This abbreviated procedure provides homogeneous material eluting at 35% acetonitrile. Purification is probably facilitated by the increased proportion of rMGSA to total protein secreted by the transfected CHO-7 cells. The yield of the purified rMGSA from the initial 5 liters of medium is approximately 60 tzg (240 ~g/20 liters), based on the relative quantitation of the integrated HPLC peak in comparison with an insulin standard. This quantity of material is much greater than previous recoveries of material from the Hs294T conditioned medium, which also requires a much more labor-intensive purification procedure. SDS-PAGE (Fig. 2) of the rMGSA suggests a molecular weight of about 8000. This is in apparent contrast to previously reported 13,000 j and 16,000 values for MGSA from the Hs294T cells. This can be explained, however, by the use of different molecular weight standards. The previously ascribed weight of 13,000 is probably incorrect based on the recent use of molecular weight standards better suited for the determination of molecular weights less than 14K. The nature and relationship of the previously reported 16K moiety to the tower molecular weight MGSA is currently under investigation. Characterization of Recombinant Melanoma Growth Stimulatory Activity
Amino-Terminal Sequencing. The purified recombinant MGSA is electrophoresed on a 15% SDS-polyacrylamide minigel. The gel is removed from the apparatus, soaked in blotting buffer (24 mM Tris base, 192 mM glycine, 15% methanol), and the protein is electrophoretically transferred
PURIFICATIONOF MGSA
[36]
379
3 m
2918.4-
14.36.23-
MRAALSAAPSNPRLLRVALLLLLVAAGRRAAG
10
20
30
ASVATELRX . ,
. . , ,
. ,
, .
.
, .
,
,
. ,
.
ASVATELRCQCLQTLQGIHPKNIQ 40
50
SVNVKSPGPHCAQTEVIATLKNGR 60
_
70
80
KACLNPASPIVKKIIEKMLNSDKSN 9O 100 FIG. 2. Purified recombinant MGSA. MGSA eluting at 35% acetonitrile from the Vydac C18 column was analyzed by SDS-polyacrylamide gel electrophoresis (12-18% linear polyacrylamide gradient) and stained with Coomassie blue. The positions of the molecular weight markers are shown at left.
for 2 hr at 170 V onto a sheet of poly(vinyl difluoride) (PVDF) (lmmobilon, Millipore, Bedford, MA) presoaked in 100% methanol for 1 min and in blotting buffer for 15 rain. The transblotted protein is stained with 0.1% Coomassie blue R-250 in 50% methanol, 10% acetic acid for 1-2 min, destained with 50% methanol, 10% acetic acid (v/v), and air-dried, l0 A 1-mm PVDF strip with the stained protein band is packed into the center of a sequencing column and sandwiched between 1 cm of controlled pore glass and C~8 reversed-phase packing. The protein can be sequenced by Edman degradation on a liquidl0 p. Matsudaira, J. Biol. Chem. 262, 10035 (1987).
380
OTHER GROWTH FACTORS AND GROWTH INHIBITORS
[36]
phase sequencer. Identical N-terminal sequences are found for MGSA from both Hs294T cells 3 and the transfected CHO-7 cells. Immunoprecipitation. Additional structural similarity is seen by the precipitation of natural and recombinant MGSA using monoclonal and polyclonal antibodies to MGSA. 11"12The FB2AH7 monoclonal antibody was developed against Hs294T-derived MGSA and has been used in several applications, namely, immunohistochemistry and immunocytochemistry, 13 enzyme-linked immunoabsorbent assay, 9 and immunoaffinity chromatography. 14 125I-Labeled rMGSA 15 [100,000 counts/min (cpm)] is incubated overnight with 0.5 ml FB2AH7 antibody in an Eppendorf tube at 4 °. Goat anti-mouse IgM (0.5 mg/0.5 ml) is added and the incubation proceeds at 25 ° for 2 hr. The precipitate is pelleted by microcentrifugation for 15 min. The pellets are washed 3 times with PBS, 0.1% Triton X-100. The pellet is resuspended in 20/xl of 2 × Laemmli electrophoresis sample buffer and electrophoresed on a 7.5-15% linear polyacrylamide gel. The gel is removed from the apparatus and stained overnight with Coomassie blue R-250. The next day the gel is destained, dried, and subjected to autoradiography using Kodak XAR-5 film (Rochester, NY). Results (Fig. 3) indicate the FB2AH7 monoclonal antibody recognizes the J25I-labeled rMGSA moiety and can be used for its detection and/or precipitation in future experiments. The higher molecular weight materials observed in the 125I-labeled rMGSA (Fig. 3, Lane 1) provide an example of the tendency of MGSA to aggregate at neutral pH.13 This aggregation is noted more frequently with iodinated MGSA. At the present time it is not known if this aggregation actually occurs more frequently in the iodinated preparations, or whether it is simply observed more often because of the greater sensitivity of the autoradiographic techniques. In analogous experiments, a polyclonal antiserum 12 raised in rabbits against an MGSA-specific oligopeptide can be utilized to immunoprecipitate MGSA from the serum-free conditioned medium of metabolically 35Slabeled CHO clone 7 cells and Hs294T cells. Cells are grown to about 70% confluency in 6-well plates. The monolayers are washed twice with PBS and once with minimal medium and then incubated overnight at 37° in 1.5 ml of serum-free medium lacking cysteine and methionine, or phosphate or sulfate (GIBCO Laboratories, Grand Island, NY). The medium is then supplemented with 100 ~Ci/ml of L-[35S]methionine. After overnight incuii D. H. Lawson, H. G. Thomas, R. G. B. Roy, D. J. Gordon, R. K. Chawla, D. W. Nixon, and A. Richmond, J. Cell. Biochem. 34, 169 (1987). 12 D. Wen, A. Rowland, and R. Derynck, EMBO J. 8, 1761 (1989). 13 A. Richmond and H. G. Thomas, J. Cell. Biochem. 36, 185 (1988). 14 H. G. Thomas and A. Richmond, Arch. Biochem. Biophys. 260, 719 (1988). 15 j. H. Han, H. G. Thomas, and A. Richmond, submitted for publication.
[36]
PURIFICATION OF MGSA
1
381
2
94" 67-
43-
m
25IB 18_
14- O FIG. 3. Immunoprecipitation of I25I-labeled MGSA. Radiolabeled MGSA was immunoprecipitated with the FB2AH7 monoclonal antibody and electrophoresed on a 7.5-15% linear polyacrylamide gel, stained overnight with Coomassie blue R-250, dried, and subjected to autoradiographic analysis. Lane 1 contains m25I-labeled MGSA; lane 2 contains 1251-1abeled MGSA immunoprecipitated by the FB2AH7 antibody.
bation the conditioned medium is collected and clarified by centrifugation, and the protease inhibitor phenylmethylsulfonyl fluoride (PMSF) is added to the medium to a 1 m M final concentration. Immunoprecipitations are carried out as follows. The samples are pretreated with 10/zl of normal rabbit serum and 20 ~1 of a 1 : 1 suspension of protein A-Sepharose at 4 ° for 1 hr. The protein A-Sepharose is removed by centrifugation, and the pretreated sample is reacted overnight at 4 ° with the rabbit antiserum at a 1 : 100 dilution in a 500-/xl reaction mixture, to which 50/zl protein A-Sepharose is added. The protein A-Sepharose beads are then pelleted and washed 3 times in 0.1% Triton X-100, 0.02% SDS, 150 m M NaCI, 5 m M E D T A , and 10 units/ml of aprotinin. The washed
382
OTHER GROWTH FACTORS AND GROWTH INHIBITORS
[36]
beads are heated with 100/~l of SDS gel electrophoresis loading buffer at 100° for 5 min. The beads are removed by centrifugation, and the supernatant is loaded on a 16.5% tricine-SDS polyacrylamide gel. 16 O-Glycanase Digestion. Immunoprecipitated MGSA protein is eluted from protein A-Sepharose beads by boiling the samples for 5 rain in 60/.,1 of 0.15% SDS. The protein sample in 50/zl, containing 1 mM calcium acetate, 10 mM D-galactonolactone, 20 mM Tris-maleate, pH 6.0, is digested with neuraminidase at a final concentration of l /zl/ml for 1 hr at 37°. After digestion, SDS electrophoresis sample buffer is added, and the samples are boiled for 5 min prior to electrophoresis. Results indicate that similar molecular weight moieties are precipitated from the conditioned medium from both CHO-7 and Hs294T cells. In addition, neither subsequent neuraminidase nor neuraminidase plus Oglycanase treatment of the immunoprecipitated proteins results in a detectable SDS-PAGE mobility shift, indicating a similar lack of O-linked carbohydrates in both proteins. Since the predicted amino acid sequence does not contain a possible N-glycosylation site, 3 we have not investigated the presence of N-linked carbohydrates. Bioassay. The bioactivity of MGSA derived from CHO-7 cells is assessed by measuring the stimulation of [3H]thymidine incorporation into DNA of serum-depleted low density cultures of Hs294T human malignant melanoma cells and by determining the increase in Hs294T cell number. The incorporation of [3H]thymidine into the melanoma cells is performed as described, ~ except the assay is done in 24-well plates instead of glass vials (Wheaton). The cell number determination is performed as described. ~ MGSA is tested at concentrations from 0.06 to 60 ng/ml. The stimulation of [3H]thymidine incorporation into DNA measured after 16 hr demonstrates a 2- to 3-fold increase in response to nanogram per milliliter levels of MGSA. Determination of Hs294T cell numbers after a 3- or 6-day exposure to such levels of MGSA also demonstrates a 2- to 3-fold increase. These values closely correspond to values attained with Hs294Tderived MGSA as previously reported.l'2 Conclusions Recombinant MGSA/gro was purified from the conditioned medium of CHO-7 cells transfected with an MGSA expression vector. The large quantity of MGSA/gro produced by these cells, in comparison to that provided from natural sources, facilitated the purification. The modified purification procedure described here resulted in a large increase in recovI6 H. Schagger and G. Von Jagow, Anal. Biochem. 166, 368 (1987).
[37]
PURIFICATION
AND CLONING
OF PD-ECGF
383
ery of bioactive MGSA. Amino-terminal sequencing, SDS-PAGE, immunoprecipitation, and bioassay demonstrate the identical physical, structural, and biological properties of natural and recombinant MGSA. The results demonstrate the advantages and applicability of molecular biological techniques to problems associated with the purification, characterization, and biological investigation of growth factors, for which there are no abundant natural sources. Acknowledgments This work was supported by National Cancer Institute Grant Ca34590, Veterans Administration Merit Awards to A.R. and H.G.T., and VA Career Development Awards to R.B. and A.R. We are indebted to Martine Gould, Gary Reece, and Mike Kelleher for excellent technical assistance.
[37] P u r i f i c a t i o n , Cloning, a n d E x p r e s s i o n o f P l a t e l e t D e r i v e d E n d o t h e l i a l Cell G r o w t h F a c t o r By CARL-HENRIK HELDIN, ULF HELLMAN, FUYUKI 1SHIKAWA, an d KOHEI MIYAZONO
Introduction Human platelets are a rich source of growth regulatory proteins, the most well characterized being platelet-derived growth factor (PDGF), platelet-derived endothelial cell growth factor (PD-ECGF), and transforming growth factor/31 (TGF-/31). PDGF stimulates the proliferation mainly of connective tissue cells, and TGF-/31 inhibits the growth of most cell types. PD-ECGF is a newly discovered 45-kDa factor with distinct functional properties; it stimulates the growth of endothelial cells of large vessels and capillaries and has angiogenic activity in vivo (for a review, see Ref. 1). PD-ECGF occurs in human platelets as well as in placenta. It does not bind to heparin-Sepharose in contrast to endothelial cell mitogens of the fibroblast growth factor (FGF) family. This chapter describes the purification, sequencing, cDNA cloning, and expression of PD-ECGF.
i K. Miyazono and C.-H. Heldin, in "Peptide Growth Factors and Their Receptors, Handbook of Experimental Pharmacology" (M. B. Sporn and A. B. Roberts, eds.), Vol. 95, p. 125. Springer-Verlag, Berlin and New York, 1990.
METHODS IN ENZYMOLOGY. VOL. 198
Copyright ~9 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
PURIFICATIONOF MGSA
373
[36] P u r i f i c a t i o n a n d C h a r a c t e r i z a t i o n o f R e c o m b i n a n t Melanoma Growth Stimulating Activity B y H . GREG THOMAS, JIN H E E H A N , E D D Y BALENTIEN, RIK DERYNCK, RODOLFO BORDONI, a n d A N N RICHMOND
Introduction
Melanoma growth stimulatory activity (MGSA) was originally purified from extracts of serum-free culture medium of Hs294T human malignant melanoma cells and a variety of melanoma tumors. MGSA exerts an autocrine-like growth effect for melanoma cells, j Only recently was a method described that provided adequate amounts of purified protein for complete characterization of MGSA secreted by the Hs294T melanoma cells. 2 This factor is structurally related to various polypeptides in the/3thromboglobulin superfamily which are 8-10 kDa basic heparin-binding proteins. 2 Subsequent isolation and characterization of cDNAs revealed a predicted length of 73 amino acids proteolytically derived from a 107 residue precursor. In addition, the nucleotide sequence of the MGSA gene possesses similarity with the human "gro" gene and the PDGF-inducible gene K C . 3-5 Northern hybridization demonstrates that MGSA/gro gene expression is not restricted to melanoma cells but is found in a variety of normal and transformed cells of different origin. 3 The recently optimized procedure for the purification of MGSA from Hs294T cell culture medium required multiple processes which were laborious and time consuming. Although adequate amounts of protein were obtained for complete characterization and initial biological studies, the total yield of homogeneous material was low for the time and expense required. Therefore, a mammalian MGSA expression system has been developed in order to produce larger quantities of MGSA. The expression system utilizes a plasmid vector construct which places the MGSA gene under the control of the cytomegalovirus promoter in Chinese hamster ovarian (CHO) cells. I A. Richmond and H. G. Thomas, J. Cell. Physiol. 129, 375 (1986). 2 H. G, Thomas and A. Richmond, Mol. Cell. Endocrinol. 57, 69 (1988). 3 A. Richmond, E. Balentien, H. G. Thomas, G. Flaggs, D. E. Barton, J. Spiess, R. Bordoni, U. Francke, and R. Derynck, EMBO J. 7, 2025 (1988). 4 A. Anisowicz, L. Bardwell, and R. Sager, Proc. Natl. Acad. Sci. U.S.A. 84, 7188 (1987). 5 p. Oquendo, J. Alberta, D. Wen, J. L. Graycar, R. Derynck, and C. D. Stiles, J. Biol. Chem. 264, 4133 (1989).
METHODS IN ENZYMOLOGY, VOL. 198
Copyright 63 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
374
OTHER GROWTH FACTORS AND GROWTH INHIBITORS
[36]
We compare here the purification of MGSA to homogeneity from the Hs294T cells and from stable CHO transfectants which secrete high levels of human MGSA. The proteins obtained from these sources are compared as to size, biological activity, N-terminal amino acids, and recognition by antibodies raised against proteins derived from both cell sources, demonstrating the identical properties and activities of the proteins. Sources of Growth Factors Tissue culture materials and media are supplied as previously described. I Hs294T cells are obtained from the ATCC (Rockville, MD), and a subline is selected which grows well on serum-free Ham's F10 medium without growth factor supplements. Transfected Chinese hamster ovary cells are selected in complete medium lacking glycine, hypoxanthine, and thymidine and supplemented with 10% dialyzed fetal bovine serum as described. 6 Assay Methods for Characterizing Melanoma Growth Stimulatory Activity
[3H]ThymidineIncorporation. MGSA bioassays are performed by measuring stimulation of [3H]thymidine incorporation in serum-depleted low density cultures of Hs294T human malignant melanoma cells according to a modification of the procedures of Iio and Sirbasku 7 as previously described. 1 Cells (8000) are plated into 28 × 61 mm glass scintillation vials (Wheaton, Millville, NJ, #22528) in 2 ml of Ham's F10 medium supplemented with 10% fetal bovine serum. Twenty-four hours later, cells are washed with 2 ml of phosphate-buffered saline (PBS: 8 g NaC1, 1.15 g Na2HPO 4, 0.2 g KCI, 0.2 g KH2PO 4, 0.1 g MgCI 2 • 6H20, 0.1 g CaCI z) and placed on serum-free F10 medium containing HEPES (30 mM) and ovalbumin (I0/zg/ml). After a 24-hr incubation in serum-free medium the medium is aspirated, and aliquots of growth factor are added in a binding buffer composed of 2 ml of serum-flee FI0 with HEPES (30 mM) and ovalbumin (10/zg/ml). Eight hours later [3H]thymidine (5/xCi) is added to each vial, and the incubation is continued for an additional 16 hr. The reaction is stopped by the addition of methanol/ethanol (3 : 1). Unincorporated [3H]thymidine is removed by repeated washing with methanol, then 10 ml of Scintiverse II is added to each vial, and the radioactivity incorpo6 R. Derynck, E. Balentien, J. H. Han, H. G. Thomas, D. Wen, A. K. Samantha, C. O. Zachariae, P. R. Griffin, R. Brachmann, W. L. Wong, K. Matsushima, and A. Richmond, Biochemistry 29, 10225 (1990). 7 M. Iio and D. Sirbasku, Cold Spring Harbor Conf. Cell Proliferation 9, 751 (1982).
[36]
PURIFICATIONOF MGSA
375
rated into DNA is counted in a Beckman liquid scintillation counter. Results are compared to controls that receive only binding buffer. Cell Number Assay. MGSA bioactivity can also be assessed in a cell number assay, j Hs294T cells (8000) are seeded into glass scintillation vials as described for the [3H]thymidine assay. After 72 hr the medium is aspirated, and the cells are washed twice with PBS and placed on serumfree F10 containing HEPES (30 raM) and ovalbumin (I0/zg/ml). Fortyeight hours later the medium is aspirated, and dilutions of MGSA in a binding buffer composed of serum-free F10 with HEPES (30 mM) and ovalbumin (10 ~g/ml) are added. On the third and sixth day after growth factor additions, cells are released by brief treatment with trypsin, and the cell number is determined from an aliquot of suspended cells counted on a hemocytometer. Denaturing Electrophoresis. Sodium dodecyl sulfate (SDS) gradient gels (linear, 12-18% polyacrylamide) are prepared as described by Laemmli 8 and poured into slabs 0.75 mm thick. Samples of MGSA from various steps of the purification are brought to less than 10/xl or dryness in a Speed Vac (Savant, Hicksville, NY) or a lyophilyzer. Twice-concentrated SDS sample buffer ( - 2 0 txl) is added, and the samples are electrophoresed at 35 mA constant current. After electrophoresis the gels are fixed and stained with Coomassie blue R-250 and/or silver (Bio-Rad, Richmond, CA, silver stain kit).
Purification of Natural Melanoma Growth Stimulatory Activity
Step l: Extraction from Hs294T Conditioned Medium. Natural MGSA was initially purified from serum-flee culture medium conditioned by confluent cultures of the Hs294T human melanoma cell line as previously described. 1 The serum-free conditioned medium is collected from P-150 culture flasks using sterile technique, and the protease inhibitor Trasylol (aprotinin) (Sigma, St. Louis, MO) is added (117 kallikrein inhibitory units per liter). The medium is centrifuged at 15,000 g to remove floating cells or cellular debris, then the supernatant is ultracentrifuged (100,000 g) for 30 min to remove subcellular organelles. The resulting supernatant is lyophilized. When 20 liters of conditioned medium has been processed in this manner, the lyophilized powder is extracted with 1 N acetic acid at room temperature, dialyzed exhaustively against 0.17 N acetic acid at 4 °, lyophilized, and stored at - 8 0 °. Step 2: BioGel P-30 Chromatography. Acetic acid extracts of approximately 20 liters of lyophilized Hs294T conditioned medium are dissolved 8 U. K. Laemmli, Nature (London) 227, 680 (1972).
376
[36]
OTHER GROWTH FACTORS AND GROWTH INHIBITORS
2.5
"~
2.0
i
1.5
H
= 1.o ~
,6O
~J~
0.5..]
2O
20
40
60
Fraction Number
FIG. I. Reversed-phase HPLC of MGSA on a p~Bondapak CI8 column. Fractions eluting from the BioGei P-30 column between the 6K and 14K standards were combined, lyophilized, and dissolved in 2 ml o f 6 : 94 : 0.05 acetonitrile/water/trifluoroacetic acid. The sample was applied to a p,Bondapak CI8 column equilibrated with the same buffer. The gradient consisted o f 6-60% acetonitrile for 60 min at 25 ° with a flow rate of 1.5 ml/min. Sixty fractions were collected. Aliquots o f the fractions were used for MGSA bioassays.
in 30 ml of 1 N acetic acid and clarified by ultracentrifugation (100,000 g for 30 min). The resulting supernatant, containing approximately 200 mg of protein, is applied to a 2.5 x 90 cm BioGel P-30 column previously equilibrated with 1 N acetic acid and calibrated with 25K, 14K, and 6K polypeptides. The protein is eluted with 1 N acetic acid at a flow rate of approximately 9 ml/hr at room temperature. Absorbance is monitored at 280 nm, and 80-drop fractions are collected. Greater than 95% of the protein initially loaded onto the column elutes prior to the 25K marker. MGSA bioactivity elutes broadly and resides in two major molecular weight regions (14,000-25,000 and 5000-10,000). 9 The apparent high molecular weight (14,000-25,000) MGSA fractions are combined, and the apparent low molecular weight (5000-10,000) MGSA fractions are combined, with the pools being labeled A and B, respectively. Step 3: Initial Reversed-Phase Chromatography. Pools A and B from the BioGel P-30 column are lyophilized separately and dissolved in 1-2 ml of acetonitrile/water/trifluoroacetic acid (6 : 94 : 0.05). Each sample is injected into a/zBondapak Cj8 cartridge column (8 x 100 mm) in a Z module and eluted with a 60-min linear gradient of acetonitrile/water/ trifluoroacetic acid (6 : 94 : 0.05 to 60 : 40 : 0.05 (v/v), pH 2.2) at a flow rate of 1.5 ml/min (Fig. 1). Absorbance is monitored at 206 nm, and 1.5-ml fractions are collected. 9 A. Richmond, H. G. Thomas, and R. G. B. Roy, this series, Vol. 146, p. i 12.
[36]
PURIFICATIONOF MGSA
377
Step 4: Heparin-Sepharose Chromatography. Heparin-Sepharose chromatography has recently been utilized as an alternative to preparative SDS-PAGE for the subsequent purification of MGSA from the initial reversed-phase HPLC fractionation. 2 Bioactive fractions eluting between 31 and 38% acetonitrile are pooled, lyophilized, redissoived in 10 mM Tris, pH 7.5, and applied to a 10 x 50 mm column of heparin-Sepharose CL6B equilibrated in the same buffer. Material that does not bind to heparinSepharose is removed by continuous flow of 10 mM Tris, pH 7.5. A 200ml linear gradient of 10 mM Tris, pH 7.5, to 10 mM Tris, 1 M sodium chloride, pH 7.5, is followed by elution with 100 ml of 10 mM Tris, 2 M sodium chloride, pH 7.5, and finally elution with 100 ml of 6 M guanidine hydrochloride. Absorbance is monitored at 280 nm, at a sensitivity of 0.1 absorbance units full scale (AUFS). The flow rate is 0.5 ml/min, and 8-ml fractions are collected. Fractions are pooled based on the absorbance at 280 nm, and lyophilized aliquots are subjected to bioassay, The material eluting from 0.1 to 0.3 M sodium chloride provides the more significant stimulation in the cell number assay (-200% control) and is utilized for subsequent reversed-phase HPLC purification. Step 5: Additional Reversed-Phase Chromatography. Bioactive material from the heparin-Sepharose column is subjected to reversed-phase HPLC fractionation on a Vydac C~8 column using a linear gradient of 6 : 94 : 0.05 to 60 : 40 : 0.05 (v/v) acetonitrile/water/trifluoroacetic acid. Absorbance is monitored at 214 nm, and 2-ml fractions are collected. Aliquots of the 2-ml fractions are analyzed by SDS-PAGE and/or bioassay prior to further chromatography. The fractions are then rechromatographed on the Vydac column using a mobile phase containing trifluoroacetic acid or heptafluorobutyric acid. The linear gradients employed consist of 25 : 75 : 0.05 to 35 : 65 : 0.05 acetonitrile/water/trifluoroacetic acid or 25 : 75 : 0.05 to 45 : 55 : 0.05 acetonitrile/water/heptafluorobutyric acid. Complete resolution of the individual moieties requires an additional two or three HPLC fractionations using the above gradient containing trifluoroacetic acid but only one additional run when using heptafluorobutyric acid. These modifications significantly increase the yield of homogeneous MGSA from less than 1/~g using preparative SDS-PAGE to 5-10/zg when using these modifications. Purification of Recombinant Melanoma Growth Stimulating Activity The purification of recombinant MGSA (rMGSA) from the CHO clone 7 cells utilizes many of the procedures previously mentioned.
Step 1: Extraction from CHO Clone 7 Conditioned Medium. Five liters
378
O T H E R G R O W T H FACTORS A N D G R O W T H I N H I B I T O R S
[36]
of serum-free conditioned medium from the CHO clone 7 cells transfected with the expression plasmid pCMV-M236 is concentrated by lyophilization, prior to dialysis against 0.17 N acetic acid. Step 2: BioGel P-30 Chromatography. The concentrate from Step 1 is then subjected to BioGel P-30 chromatography as previously described. Step 3: Heparin-Sepharose Chromatography. Fractions eluting from the BioGel P-30 column between the ribonuclease A and insulin markers (13.7K and 6K, respectively) are pooled, lyophilized, and subjected to heparin-Sepharose affinity chromatography as previously described. 2 Step 4: Reversed-Phase Chromatography. Fractions eluting from the heparin-Sepharose column with 0.5 M NaCI are pooled, lyophilized, and dissolved in a solution containing acetonitrile, water, and trifluoroacetic acid (6:94:0.05) (v/v). Subsequent reversed-phase HPLC is performed on a Vydac Hi-Pore C~8 column using a 60-min linear gradient from 6 to 60% acetonitrile. 2 This abbreviated procedure provides homogeneous material eluting at 35% acetonitrile. Purification is probably facilitated by the increased proportion of rMGSA to total protein secreted by the transfected CHO-7 cells. The yield of the purified rMGSA from the initial 5 liters of medium is approximately 60 tzg (240 ~g/20 liters), based on the relative quantitation of the integrated HPLC peak in comparison with an insulin standard. This quantity of material is much greater than previous recoveries of material from the Hs294T conditioned medium, which also requires a much more labor-intensive purification procedure. SDS-PAGE (Fig. 2) of the rMGSA suggests a molecular weight of about 8000. This is in apparent contrast to previously reported 13,000 j and 16,000 values for MGSA from the Hs294T cells. This can be explained, however, by the use of different molecular weight standards. The previously ascribed weight of 13,000 is probably incorrect based on the recent use of molecular weight standards better suited for the determination of molecular weights less than 14K. The nature and relationship of the previously reported 16K moiety to the tower molecular weight MGSA is currently under investigation. Characterization of Recombinant Melanoma Growth Stimulatory Activity
Amino-Terminal Sequencing. The purified recombinant MGSA is electrophoresed on a 15% SDS-polyacrylamide minigel. The gel is removed from the apparatus, soaked in blotting buffer (24 mM Tris base, 192 mM glycine, 15% methanol), and the protein is electrophoretically transferred
PURIFICATIONOF MGSA
[36]
379
3 m
2918.4-
14.36.23-
MRAALSAAPSNPRLLRVALLLLLVAAGRRAAG
10
20
30
ASVATELRX . ,
. . , ,
. ,
, .
.
, .
,
,
. ,
.
ASVATELRCQCLQTLQGIHPKNIQ 40
50
SVNVKSPGPHCAQTEVIATLKNGR 60
_
70
80
KACLNPASPIVKKIIEKMLNSDKSN 9O 100 FIG. 2. Purified recombinant MGSA. MGSA eluting at 35% acetonitrile from the Vydac C18 column was analyzed by SDS-polyacrylamide gel electrophoresis (12-18% linear polyacrylamide gradient) and stained with Coomassie blue. The positions of the molecular weight markers are shown at left.
for 2 hr at 170 V onto a sheet of poly(vinyl difluoride) (PVDF) (lmmobilon, Millipore, Bedford, MA) presoaked in 100% methanol for 1 min and in blotting buffer for 15 rain. The transblotted protein is stained with 0.1% Coomassie blue R-250 in 50% methanol, 10% acetic acid for 1-2 min, destained with 50% methanol, 10% acetic acid (v/v), and air-dried, l0 A 1-mm PVDF strip with the stained protein band is packed into the center of a sequencing column and sandwiched between 1 cm of controlled pore glass and C~8 reversed-phase packing. The protein can be sequenced by Edman degradation on a liquidl0 p. Matsudaira, J. Biol. Chem. 262, 10035 (1987).
380
OTHER GROWTH FACTORS AND GROWTH INHIBITORS
[36]
phase sequencer. Identical N-terminal sequences are found for MGSA from both Hs294T cells 3 and the transfected CHO-7 cells. Immunoprecipitation. Additional structural similarity is seen by the precipitation of natural and recombinant MGSA using monoclonal and polyclonal antibodies to MGSA. 11"12The FB2AH7 monoclonal antibody was developed against Hs294T-derived MGSA and has been used in several applications, namely, immunohistochemistry and immunocytochemistry, 13 enzyme-linked immunoabsorbent assay, 9 and immunoaffinity chromatography. 14 125I-Labeled rMGSA 15 [100,000 counts/min (cpm)] is incubated overnight with 0.5 ml FB2AH7 antibody in an Eppendorf tube at 4 °. Goat anti-mouse IgM (0.5 mg/0.5 ml) is added and the incubation proceeds at 25 ° for 2 hr. The precipitate is pelleted by microcentrifugation for 15 min. The pellets are washed 3 times with PBS, 0.1% Triton X-100. The pellet is resuspended in 20/xl of 2 × Laemmli electrophoresis sample buffer and electrophoresed on a 7.5-15% linear polyacrylamide gel. The gel is removed from the apparatus and stained overnight with Coomassie blue R-250. The next day the gel is destained, dried, and subjected to autoradiography using Kodak XAR-5 film (Rochester, NY). Results (Fig. 3) indicate the FB2AH7 monoclonal antibody recognizes the J25I-labeled rMGSA moiety and can be used for its detection and/or precipitation in future experiments. The higher molecular weight materials observed in the 125I-labeled rMGSA (Fig. 3, Lane 1) provide an example of the tendency of MGSA to aggregate at neutral pH.13 This aggregation is noted more frequently with iodinated MGSA. At the present time it is not known if this aggregation actually occurs more frequently in the iodinated preparations, or whether it is simply observed more often because of the greater sensitivity of the autoradiographic techniques. In analogous experiments, a polyclonal antiserum 12 raised in rabbits against an MGSA-specific oligopeptide can be utilized to immunoprecipitate MGSA from the serum-free conditioned medium of metabolically 35Slabeled CHO clone 7 cells and Hs294T cells. Cells are grown to about 70% confluency in 6-well plates. The monolayers are washed twice with PBS and once with minimal medium and then incubated overnight at 37° in 1.5 ml of serum-free medium lacking cysteine and methionine, or phosphate or sulfate (GIBCO Laboratories, Grand Island, NY). The medium is then supplemented with 100 ~Ci/ml of L-[35S]methionine. After overnight incuii D. H. Lawson, H. G. Thomas, R. G. B. Roy, D. J. Gordon, R. K. Chawla, D. W. Nixon, and A. Richmond, J. Cell. Biochem. 34, 169 (1987). 12 D. Wen, A. Rowland, and R. Derynck, EMBO J. 8, 1761 (1989). 13 A. Richmond and H. G. Thomas, J. Cell. Biochem. 36, 185 (1988). 14 H. G. Thomas and A. Richmond, Arch. Biochem. Biophys. 260, 719 (1988). 15 j. H. Han, H. G. Thomas, and A. Richmond, submitted for publication.
[36]
PURIFICATION OF MGSA
1
381
2
94" 67-
43-
m
25IB 18_
14- O FIG. 3. Immunoprecipitation of I25I-labeled MGSA. Radiolabeled MGSA was immunoprecipitated with the FB2AH7 monoclonal antibody and electrophoresed on a 7.5-15% linear polyacrylamide gel, stained overnight with Coomassie blue R-250, dried, and subjected to autoradiographic analysis. Lane 1 contains m25I-labeled MGSA; lane 2 contains 1251-1abeled MGSA immunoprecipitated by the FB2AH7 antibody.
bation the conditioned medium is collected and clarified by centrifugation, and the protease inhibitor phenylmethylsulfonyl fluoride (PMSF) is added to the medium to a 1 m M final concentration. Immunoprecipitations are carried out as follows. The samples are pretreated with 10/zl of normal rabbit serum and 20 ~1 of a 1 : 1 suspension of protein A-Sepharose at 4 ° for 1 hr. The protein A-Sepharose is removed by centrifugation, and the pretreated sample is reacted overnight at 4 ° with the rabbit antiserum at a 1 : 100 dilution in a 500-/xl reaction mixture, to which 50/zl protein A-Sepharose is added. The protein A-Sepharose beads are then pelleted and washed 3 times in 0.1% Triton X-100, 0.02% SDS, 150 m M NaCI, 5 m M E D T A , and 10 units/ml of aprotinin. The washed
382
OTHER GROWTH FACTORS AND GROWTH INHIBITORS
[36]
beads are heated with 100/~l of SDS gel electrophoresis loading buffer at 100° for 5 min. The beads are removed by centrifugation, and the supernatant is loaded on a 16.5% tricine-SDS polyacrylamide gel. 16 O-Glycanase Digestion. Immunoprecipitated MGSA protein is eluted from protein A-Sepharose beads by boiling the samples for 5 rain in 60/.,1 of 0.15% SDS. The protein sample in 50/zl, containing 1 mM calcium acetate, 10 mM D-galactonolactone, 20 mM Tris-maleate, pH 6.0, is digested with neuraminidase at a final concentration of l /zl/ml for 1 hr at 37°. After digestion, SDS electrophoresis sample buffer is added, and the samples are boiled for 5 min prior to electrophoresis. Results indicate that similar molecular weight moieties are precipitated from the conditioned medium from both CHO-7 and Hs294T cells. In addition, neither subsequent neuraminidase nor neuraminidase plus Oglycanase treatment of the immunoprecipitated proteins results in a detectable SDS-PAGE mobility shift, indicating a similar lack of O-linked carbohydrates in both proteins. Since the predicted amino acid sequence does not contain a possible N-glycosylation site, 3 we have not investigated the presence of N-linked carbohydrates. Bioassay. The bioactivity of MGSA derived from CHO-7 cells is assessed by measuring the stimulation of [3H]thymidine incorporation into DNA of serum-depleted low density cultures of Hs294T human malignant melanoma cells and by determining the increase in Hs294T cell number. The incorporation of [3H]thymidine into the melanoma cells is performed as described, ~ except the assay is done in 24-well plates instead of glass vials (Wheaton). The cell number determination is performed as described. ~ MGSA is tested at concentrations from 0.06 to 60 ng/ml. The stimulation of [3H]thymidine incorporation into DNA measured after 16 hr demonstrates a 2- to 3-fold increase in response to nanogram per milliliter levels of MGSA. Determination of Hs294T cell numbers after a 3- or 6-day exposure to such levels of MGSA also demonstrates a 2- to 3-fold increase. These values closely correspond to values attained with Hs294Tderived MGSA as previously reported.l'2 Conclusions Recombinant MGSA/gro was purified from the conditioned medium of CHO-7 cells transfected with an MGSA expression vector. The large quantity of MGSA/gro produced by these cells, in comparison to that provided from natural sources, facilitated the purification. The modified purification procedure described here resulted in a large increase in recovI6 H. Schagger and G. Von Jagow, Anal. Biochem. 166, 368 (1987).
[37]
PURIFICATION
AND CLONING
OF PD-ECGF
383
ery of bioactive MGSA. Amino-terminal sequencing, SDS-PAGE, immunoprecipitation, and bioassay demonstrate the identical physical, structural, and biological properties of natural and recombinant MGSA. The results demonstrate the advantages and applicability of molecular biological techniques to problems associated with the purification, characterization, and biological investigation of growth factors, for which there are no abundant natural sources. Acknowledgments This work was supported by National Cancer Institute Grant Ca34590, Veterans Administration Merit Awards to A.R. and H.G.T., and VA Career Development Awards to R.B. and A.R. We are indebted to Martine Gould, Gary Reece, and Mike Kelleher for excellent technical assistance.
[37] P u r i f i c a t i o n , Cloning, a n d E x p r e s s i o n o f P l a t e l e t D e r i v e d E n d o t h e l i a l Cell G r o w t h F a c t o r By CARL-HENRIK HELDIN, ULF HELLMAN, FUYUKI 1SHIKAWA, an d KOHEI MIYAZONO
Introduction Human platelets are a rich source of growth regulatory proteins, the most well characterized being platelet-derived growth factor (PDGF), platelet-derived endothelial cell growth factor (PD-ECGF), and transforming growth factor/31 (TGF-/31). PDGF stimulates the proliferation mainly of connective tissue cells, and TGF-/31 inhibits the growth of most cell types. PD-ECGF is a newly discovered 45-kDa factor with distinct functional properties; it stimulates the growth of endothelial cells of large vessels and capillaries and has angiogenic activity in vivo (for a review, see Ref. 1). PD-ECGF occurs in human platelets as well as in placenta. It does not bind to heparin-Sepharose in contrast to endothelial cell mitogens of the fibroblast growth factor (FGF) family. This chapter describes the purification, sequencing, cDNA cloning, and expression of PD-ECGF.
i K. Miyazono and C.-H. Heldin, in "Peptide Growth Factors and Their Receptors, Handbook of Experimental Pharmacology" (M. B. Sporn and A. B. Roberts, eds.), Vol. 95, p. 125. Springer-Verlag, Berlin and New York, 1990.
METHODS IN ENZYMOLOGY. VOL. 198
Copyright ~9 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
