Cell, Vol. 63, 195-201,

October

5, 1990, Copyright

0 1990 by Cell Press

Identification, Purification, and Biological Characterization of Hematopoietic Stem Cell Factor from Buffalo Rat Liver-Conditioned Medium Krisztina M. Zsebo, Jette Wypych, Ian K. McNiece, Hsieng S. Lu, Kent A. Smith, Subash B. Karkare, Raj K. Sachdev, Victoria N. Vuschenkoff, Neal C. Birkett, Lee Ft. Williams, Vasuki N. Satyagal, Weifong Tung, Robert A. Bosselman, Elizabeth A. Mendiaz, and Keith E. Langley AMGEN Inc. AMGEN Center Thousand Oaks, California 91320

Summary We have identified a novel growth factor, stem cell factor (SCF), for primitive hematopoietic progenitors based on its activity on bone marrow cells derived from mice treated with 5fluorouracil. The protein was isolated from the medium conditioned by Buffalo rat liver cells. It is heavily glycosylated, with both N-linked and O-linked carbohydrate. Amino acid sequence following removal of N-terminal pyroglutamate is presented. The protein has potent synergistic activities in semisolid bone marrow cultures in conjunction with colony-stimulating factors. It is also a growth factor for mast cells. In two companion papers, we present the sequences of partial SCF cDNAs, identify SCF as a c-kit ligand, and map the SCF gene to the SI locus of the mouse. Introduction Several hematopoietic growth factors have been identified by their ability to stimulate bone marrow-derived colony growth in semisolid cultures (Metcalf, 1985; Clark and Kamen, 1987). The cellular targets of growth factors such as granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-C!%), colony-stimulating factor 1 (CSF-l), and interleukin 3 (IL-3) or multi-CSF are rapidly proliferating myeloid-committed progenitors in the bone marrow (Hodgson et al., 1982; Van Zant, 1984; Zsebo et al., 1988). Some colony-stimulating factors have proven efficacious in hematopoietic deficiency states of anemia (Eschbach et al., 1987) and neutropenia (Gabrilove et al., 1988). The primitive pluripotent hematopoietic stem cells, capable of long-term reconstitution in a bone marrow transplant, are more quiescent and slowly cycling (Lerner and Harrison, 1990). These cells do not directly form colonies in agar (Andrews et al., 1989) but lead to multiple colonies of mixed lineage after culture with bone marrow stromal cells. Growth factors for early progenitors may be useful for enhancing the success of gene transfer into stem cells as well as treating disorders resulting in pancytopenia. To study stem cells, which represent less than 0.1% of total bone marrow (Spangrude et al., 1988) in vivo or in vitro enrichment techniques are necessary. By utilizing in

vivo enrichment for slowly proliferating cells in the marrow, we have identified a growth factor that acts on these early cells. The factor is present in the medium conditioned by Buffalo rat liver (BRL9A) cells. In this paper we describe identification, purification, and biochemical and biological characterization of the isolated rat protein. In the accompanying papers (Martin et al., 1990; Zsebo et al., 1990) we describe the isolation and sequence of rat and human partial cDNAs as well as studies indicating that the factor is the product of the steel (S/) locus of the mouse and is a ligand for the tyrosine kinase-type receptor c-kit. The SI gene product is known to be necessary for in vitro (Dexter and Moore, 1977; Zsebo et al., 1990) and in vivo (Ploemacher et al., 1988; Wright and Lorimore, 1987) stem cell survival. Hematopoietic stem cells depend on the receptor-ligand interaction mediated by c-kit for proliferation (Geissler and Russell, 1983; Geissler et al., 1988). Hence the BRL-SA-derived protein is named stem cell factor (SCF). Results

and Discussion

Identification of SCF Activity In vivo administration of 5fluorouracil (5-FU) to mice destroys rapidly proliferating hematopoietic progenitors but spares the more quiescent primitive stem cells capable of long-term repopulation (Hodgson and Bradley, 1979; Lerner and Harrison, 1990). When mice are treated with 5-FU at a dose of 150 mglkg, there is a significant decrease in overall bone marrow cellularity, and enrichment for primitive hematopoietic stem cells by an order of magnitude can be achieved. With 2 day post 5-FU bone marrow cells as the target population, GM-CSF (Figure l), IL-3, and G-CSF (Zsebo et al., 1988) fail to stimulate colony formation in agar. Therefore, this population is devoid of cells capable of direct colony formation. In contrast to the case with the other factors, the bone marrow targets of rat SCF are not abolished by in vivo 5FU treatment (Figure 1). Using an assay system that measures high proliferative potential colony-forming cells (HPP-CFC) present in post 5FU bone marrow (Bradley and Hodgson, 1979; McNiece et al., 1990) steps enabling purification of SCF from BRL-3Aconditioned medium were developed. In addition, we found that the murine mast cell line MC/9 (Nabel et al., 1981; Galli et al., 1982) proliferates in response to a factor in the BRL3A medium. The mast cell activity and HPP-CFC activity cofractionated during SCF purification. BRL9A cells do not secrete interleukins 1, 2, 3, 5, 6, G-CSF, or GM-CSF as judged by the use of factor-dependent cell lines and bioassays for these factors (data not shown). Purification and Biochemical Characterization of Rat SCF To generate large amounts of conditioned medium for purification, BRL-3A cells were grown in a fermenter

Cdl 196

NORMAL

Figure 1. Normal CSF and to SCF

BM

5-FU

BM

and post 5-FU Marrow

Responses

to Murine

GM-

Normal bone marrow (EM) from C57BU6J mice, or marrow from mice treated 2 days prior with 150 mglkg 5-FU, was cultured in the presence of either murine GM-CSF (100 nglml) or purified rat SCF (100 nglml) as described in Experimental Procedures. Fourteen days later, colonies composed of at least 50 cells were scored.

perfusion culture system as described in Experimental Procedures. Rat SCF was purified from the conditioned medium by a series of steps including anion exchange chromatography, gel filtration, immobilized lectin chromatography, cation exchange chromatography, and reversephase chromatography, details of which are also described in Experimental Procedures. Figures 2A-2E show absorbance and biological activity for the column steps. The HPP-CFC assay was used to monitor all purification steps, and the MC/9 mast cell line assay was used to monitor all steps subsequent to gel filtration. Purification results are summarized in Table 1. Calibration of the gel filtration column with molecular weight standards indicated apparent molecular weight for the eluting active material of 70,000-90,000. For the final purification step, i.e., the reverse-phase chromatography, it can be seen that the presence of material migrating broadly over the range of M, 28,000-35,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) correlates with the presence of activity in the fractions (Figures 2E and

B

FRACTION NUMBER Figure

2. Purification

of SCF From

BRL-BA-Conditioned

Medium

Steps were carried out as described in Experimental Procedures. (A) DEAE-cellulose anion exchange. The results for one of the several runs are shown. Fractions collected during sample application and wash are not shown; no biological activity was detected in these fractions. The NaCl gradient (see Experimental Procedures) was started at fraction 1. Fractions 67-111 were pooled. (B) Ultrogel AcA 54 gel filtration. Fractions 70-112 were pooled. The apparent split peak of activity is artifactual due to inhibitors eluting at the major absorbance peak. (C)Wheat germ agglutinin-agarose. One of the two runs carried out is represented. Fraction 1 corresponds to the start of sample application. Elution with N-acetyl-o-glucosamine-containing buffer was begun at fraction 161. Fractions 211-225 were pooled. (D) S-Sepharose cation exchange. Fraction 1 corresponds to the start of sample application. The NaCl gradient was started at fraction 50. Fractions 4-40 were pooled. (E) Cq reverse-phase chromatography. The first of the two runs referred to in Experimental Procedures is represented. Fractions collected during sample application and wash are not shown. Fraction 1 corresponds to the start of the elution gradient. Fractions 4-6 were pooled. (F) Analysis of fractions from the Cq column (E) by SDS-PAGE with silver staining. Lane A, load applied to column (75 ~1); lane B, runthrough plus wash (75 ul); lanes 4-9, aliquots (60 ul) of fractions with the corresponding numbers. The numbers (~10~) at left represent migration positions of markers having the indicated M, values. The HPP-CFC activity was not measured for the column runs of (D) and (E) because we had found in previous equivalent runs that the HPP-CFC and MC/9 activities coeluted.

Purification 197

Table

of Stem Cell Factor

1. Summary

of Purification

noncovalently associated dimer under nondenaturing conditions. Recombinant SCFs recovered from Escherichia coli in active and unglycosylated form (Martin et al., 1990) also exist as noncovalently associated dimers in solution, as judged by gel filtration and sedimentation velocity analyses (unpublished data). Since SCF has been identified as a ligand for the receptor c-kit (Zsebo et al., 1990) it is of particular interest that SCF appears to be dimeric. c-kit bears significant structural homology to the CSF-l/PDGF receptor kinase family (Quiy et al., 1988). Both CSF-1 (Das and Stanley, 1982) and PDGF (Johnsson et al., 1982) exist as covalently associated dimers. The apparent heterogeneity of the SCF by SDS-PAGE is largely due to extensive and heterogeneous glycosylation. Figure 3 (lanes 3-9) shows the results of treatments of 1251-labeled SCF with a variety of glycosidases, singly and in combination, followed by SDS-PAGE and autoradiography. Treatment with neuraminidase, which removes sialic acid, led to an overall increase in mobility (lane 4). Treatment with both neuraminidase and 0-glycanase, which removes certain O-linked carbohydrates (Umemoto et al., 1977) led to a further increase in overall mobility (lane 5). Treatment with N-glycanase, which removes both complex and high mannose N-linked carbohydrate (Tarentino et al., 1985), led to a sharper band with an apparent M, of about 28,000 (lane 8). Treatment with endoglycosidase F, which acts similarly to N-glycanase (Elder and Alexander, 1982), gave a result indistinguishable from that shown in lane 8 for N-glycanase. Treatment with endoglycosidase l-l, which removes high mannose and certain hybrid-type N-linked carbohydrate (Tarentino et al., 1978) was without effect (data not shown). Treatment with N-glycanase plus neuraminidase (lane 7) led to a fairly sharp band with an apparent M, of about 23,000. Finally, treatment with N-glycanase, neuraminidase, and O-glycanase together (lane 8) led to a doublet. The fastest migrating band in this doublet (apparent M, about 18,00019,000) probably represents fully deglycosylated SCF, since amino acid sequencing data (below) in combination

of SCF

Step

Volume

Conditioned Medium DEAE-Celluloseb AcA 54 Wheat Germ Agglutinin-AgaroseC S-Sepharose C4 Resin0

335,700 2,900 550 375 540d 57

(ml)

Total Protein (mg)a 13,475 2,164 1,513 431 10 0.25-0.406’

a Determined by the method of Bradford (1976) except where indicated otherwise. b Values given represent sums of the values for the different batches described in the text. c As described in Experimental Procedures, precipitated material that appeared during dialysis of this sample in preparation for S-Sepharose chromatography was removed by centrifugation. The sample after centrifugation (460 ml) contained 264 mg of total protein. * Only 450 ml of this material was used for the following step. B Combination of the active gradient fractions from the two Cq columns run in sequence as described. r Estimate, based on intensity of silver staining after SDS-PAGE and on quantitative amino acid composition analysis (data not shown).

2F; SDS-PAGE of the pooled materials from reversephase chromatography is shown in Figure 3, lanes 1 and 2); this correlation suggests that the broadly migrating material on SDS-PAGE represents SCF. Several additional lines of evidence support this conclusion. Upon elution and assay of material from gel slices obtained after SDS-PAGE of the purified material, we recover biological activity from the M, 28,000-35,000 region of the gel (data not shown). Furthermore, amino acid sequence information obtained for the purified material (below; Martin et al., 1990) has led to the isolation of partial cDNAs encoding these sequences, and recombinant protein possesses the same in vitro biological activities as the natural protein (Martin et al., 1990). The difference in the molecular weight estimation of SCF by gel filtration and that by SDS-PAGE suggests that the molecule may exist as a

Figure 3. SDS-PAGE of Results of Deglycosylation

9z466.2-

21.5 -

2

SCF

and

Lane 1, SDS-PAGE, with silver staining, of aliquot from pool of the first Cq column referred to in Experimental Procedures and Figure 2E; lane 2. as lane 1, with an aliquot from pool of second Ca column referred to in Experimental Procedures. Lanes 3-9, results of treatments of 1251-labeled SCF with various glycosidases, followed by SDS-PAGE and autoradiography. Lanes 3 and 9, 12sl-labeled SCF without any glycosidase treatment. Lanes 4-6, ‘*sl-labeled SCF treated with glycosidases, as follows: lane 4. neuraminidase; lane 5, neuraminidase and 0-glycanase; lane 6, N-glycanase; lane 7, neuraminidase and N-glycanase; lane 6, neuraminidase, C-glycanase, and Nglycanase. The numbers (~10s) to the left of gel lanes represent migration positions of markers having the indicated M, values.

42.7-

1

Purified

3

4

5

6

7

8

9

Cell 196

with amino acid sequence deduced from nucleotide sequence of the partial cDNA (Martin et al., 1990) indicate that the SCF, as isolated, represents a polypeptide of 164-165 amino acids with a calculated M, of about 18,500. The results depicted in Figure 3 allow the following additional conclusions: both N-linked and O-linked carbohydrates are present on SCF; most of the N-linked carbohydrate is of the complex type; and sialic acid is present, with at least some of it being part of the O-linked moieties. The giycosylation of SCF is consistent with the finding that it binds to immobilized wheat germ agglutinin (Figure 2C). SCF is very acidic, as indicated by the lack of binding to S-Sepharose resin at pH 4.2 (Figure 2D); this is consistent with the presence of sialic acid residues in the carbohydrate component. N-Termlnal Amino Acid Sequence of Purified SCF Several attempts to sequence directly SCF protein from the N-terminus were unsuccessful. in addition, purified SCF was partially deglycosyiated with N-glycanase and subjected to SDS-PAGE followed by eiectroblotting onto poiyvinyidifluoride (WDF) membrane. Bands on the membrane corresponding to the SCF forms were excised and subjected to N-terminal sequence analysis. Again, no signals were detected. Thus SCF appeared to be blocked to Edman degradation at the N-terminus. When SCF was incubated with calf liver pyrogiutamate aminopeptidase and the reaction mixture directly sequenced, a major sequence was clearly identified as Glu-ile-Cys-Arg-Asn-Pro-Val-ThrAspAsn-Val-LysAsp Pyroglutamate aminopeptidase removes N-terminal pyroglutamate from proteins or peptides (Podeil and Abraham, 1978), exposing an unblocked N-terminus. Therefore, the N-terminal amino acid of SCF is pyroglutamic acid, preceding the amino acid sequence shown above. Furthermore, the entire amino acid sequence of the isolated rat SCF has been determined by protein methods (H. S. L., submitted) and it is clear that the sequence is unique; the poiypeptide terminates at amino acid 164 or 165; and the entire sequence is encoded within the partial rat cDNA that is described in the following paper (Martin et al., 1990). However, it is likely that the open reading frame for the the rat SCF gene extends past the codon for amino acid 165. Thus the natural rat SCF polypeptide as isolated may be the product of posttranslational processing. This is especially likely from comparison with the amino acid sequence encoded in the complete open reading frame of the human cDNA reported in the following paper (Martin et al., 1990). The deduced human amino acid sequence includes 248 amino acids subsequent to the putative leader sequence, with a putative transmembrane hydrophobic domain that would correspond to amino acids 190-212 of the postleader sequence. In any case, both the isolated natural rat SCF and recombinant-derived rat SCFls4 are biologically active in the variety of ways outlined in this paper and the following two papers. Biological Activity of Purified Rat SCF To explore the effects of purified SCF on bone marrow cells, experiments were carried out with total marrow, mar-

Table 2. Effect

of SCF on Different Colony

Growth

Factorsa

SCF hlL-6 hlL-16 mlL-3 hlL-16 + hlL-6 SCF + hlL-6 SCF + hlL-16 SCF + mlL-3

Bone Marrow

Populations

Formation

Total Bone Marrowb

Lineage-Negative Bone MarrowC

post 5FU Bone Marrowd

26 1 0 10 0 29 29 45

3+1 0 0 5?2 0 15 f 3 5t1 11 % 4

34 + 0 l&l 2+2 0 43 k 51 + 28 +

+ 5 +o + 3 + 3 f 6 + 4

1

8 10 6

a The designations h and m refer to human and murine, respectively. Growth factors were used at the following concentrations: SCF (purified as described in Experimental Procedures), 100 rig/ml; hlL-6, 20 nglml; hlL-15, 20 rig/ml; mlL-3, 100 U/ml. b Total bone marrow cells were obtained from C57BU6 x DBAlP Fl mice and plated as described in Experimental Procedures, at a density of 5 x lo4 cells pet ml. c Lineage-negative bone marrow cells were obtained as above except that prior to plating, cells bearing lineage markers were removed with a panel of rat antibodies to mature myeloid and lymphoid cell antigens including 714, 14.6, YW 13.1 .l, L3T4, Lyt 2, and Thy 1.2 as described in Experimental Procedures. Cells were plated at a density of 10s cells per ml. d C57BU6J mice were injected I.V. with 5-FU at a dose of 150 mglkg. and bone marrow was harvested 2 days later. The cells were washed and plated at a nucleated cell density of 2 x 10s cells per ml as described in Experimental Procedures.

row depleted of mature myeloid and lymphoid elements (lineage-negative marrow), and marrow from 5-FU-treated mice. The latter two are enriched for primitive progenitors (Lerner and Harrison, 1990; Bertoncello et al., 1989). In cultures of total marrow, SCF alone stimulates colony formation (Table 2). The colonies consist of granuiocytes and monocytes, as determined by specific esterase staining, and megakaryocytes, as determined by staining with acetyicholinesterase (data not shown). In cultures of lineage-negative marrow, SCF alone has little effect but acts synergistically with hlL-6 (Table 2). The lineage-negative cells are enriched 25-fold in colonyforming cells in comparison to total bone marrow (Bertoncello et al., 1989). The findings for normal and lineagenegative marrow, taken together, suggest that SCF does not act directly on colony-forming ceils. The increased colony number and colony size resulting from combinations of SCF and other growth factors may reflect production by accessory cells of secondary factors that lead to increased proliferation of the colony-forming cells. Alternatively, SCF may lead to expansion or activation of precolony-forming cells such that they become responsive to colony-stimulating factors. In cultures of 2 day post 5-FU bone marrow, SCF stimulates colony formation and is synergistic in this regard with hlL-6 and hll-16 (Table 2). Cells capable of long-term reconstitution are present in 2 day post 5-FU marrow (Lerner and Harrison, 1990) whereas the cells capable of proliferation in response to colony-stimulating factors are absent (Figure 1; Zsebo et al., 1988). hlL-6 (Wong et al., 1988) and IL-l 6 (Zsebo et al., 1988) act on primitive hematopoietic progenitors. hll-6, hll-lj$ and mlL-3 alone do not

Purification 199

of Stem Cell

Factor

stimulate colony formation from the 2 day post 5FU marrow. The need for high numbers of plated cells in order to elicit colony formation by 2 day post 5-FU marrow may be explained by the presence of mature myeloid elements, which were apparently past the phase of rapid proliferation at the time of 5-FU administration. Purified stem cells (Spangrude et al., 1988) from normal marrow do not form colonies in agar in response to colony-stimulating factors. The bone marrow derived from animals in the recovery phase following myeloablative (i.e., 5-FU) treatment shows more SCF-induced colony formation than normal marrow depleted of mature cells, perhaps as a result of in vivo priming. The effects on various bone marrow populations described in this and the following papers indicate that SCFlw acts on early hematopoietic progenitors. Purification techniques exist for hematopoietic stem cells (Bertoncello et al., 1985; Mulder and Visser, 1987; Spangrude et al., 1988; Ploemacher and Brons, 1989) and experiments to assess the effects of SCF on such cell populations are in progress. The stem cell activity of SCF is further implicated by our finding that it is a product of the SI locus of the mouse (Zsebo et al., 1990) which is required for stem cell survival (Dexter and Moore, 1977; Zsebo et al., 1990). SCF is normally produced by marrow stromal cells (I. K. M., submitted). Stromal cells from SI mutant mice do not support CFU& in vitro (Zsebo et al., 1990). In addition, as mentioned above, SCF is a ligand for the receptor c-kit (Zsebo et al., 1990) which is present on hematopoietic stem cells and is required for their proliferation (Geissler and Russell, 1983). The action of SCF in stimulating proliferation of MC/9 cells (Figure 2; Martin et al., 1990) suggests that SCF is also involved in the development of mast cells. This is consistent with previous findings that both SI and dominant white spotting (WJ mutant mice (the latter are defective in the receptor c-kit; Geissler et al., 1988) exhibit deficiency in tissue mast cells (Kitamura et al., 1989). However, SCF does not stimulate mast cell degranulation as determined by measuring [3H]serotonin release from MC/9 cells using the method of Mazingue et al. (1978) (data not shown). There are certain human diseases such as aplastic anemia, Diamond Blackfan anemia, and diseases resulting in pancytopenia where defects in stem cell support and survival are thought to play a causative role. Steel (S//W) mice provide an in vivo model of genetically determined anemia caused by mutations affecting stromal support of hematopoietic stem cells (Dexter and Moore, 1977). In one of the companion papers, we show that SCF cures the anemia of S//S/d mice (Zsebo et al., 1990). A growth factor for stem cells may improve the outcome of patients undergoing bone marrow transplantation or stem cell-mediated gene replacement therapies. Experimental

Procedures

Murfne Bone Marrow Cultures All animals were purchased from Jackson Labs, Bar Harbor, ME. For experiments described in Figure 1 and Table 2, bone marrow from C57BU6J mice (either normal animals or animals treated 2 days prior with 5FU at a dose of 150 mg/kg) was plated in agar cultures contain-

ing McCoy’s complete medium, 20% fetal bovine serum (FBS), and 0.3% agar, at 2 x 10s cells per ml. The McCoy’s complete medium contained the following additions from GIBCO (supplied as 100x stock concentrations): 0.1 mM pyruvate, 0.24x essential amino acids, 0.24x nonessential amino acids. 0.027% sodium bicarbonate. 0.24x vitamins, 0.72 mM glutamine,’ ug/ml L-serine, and 12 ug/ml L-asparagine. After 14 days of incubation in a humidified atmosphere, colonies were scored. For the standard HPP-CFC assay used to monitor SCF purification, marrow from 5FU-treated BALB/c mice was used. In this case, MiaPaCa-conditioned medium (2%, as a source of CSF-1; Yunis et al., 1984) was present. Forthe experiment described in Table 2, lineage-negative bone marrow cells were obtained from C57BU8J x DBA/2 Fl mice by incubating marrow for 80 min on ice with a panel of rat antibodies including 7/4 (Hirsch and Gordon, 1983) 14.8 (Coffman, 1982) YW 13.1.1 (Watt et al., 1987) L3T4 (Becton Dickinson, Mountain View, CA), Lyt 2 (Becton Dickinson), and Thy 1.2 (Becton Dickinson). The cells were washed three times, resuspended in 8 ml of phosphate-buffered saline plus 2% FBS and 1.2 ml of magnetic beads coupled to sheep anti-rat IgG (Dynal Inc.). and incubated at 4OC for 80 min with constant mixing. Cells bound to the beads were removed by magnetic separation (Magnetic Particle Concentrator, Dynal Inc.) and discarded. The antibody negative cells (lineage negative) were washed and plated at a density of 103 cells per ml as described above. MC19 Cell Proliferation MC19 cells (Nabel et al., 1981) were subcloned to reduce the incidence of factor-independent growth and used to monitor the purification of SCF from BRL-3Aconditioned medium. MC/9 cells were cultured according to the American Type Culture Collection (ATCC; Bethesda, MD) CRL 8306 protocol. The cells were starved without a source of growth factor for 24 hr prior to plating and assayed for proliferation essentially as described (Nabel et al., 1981). Briefly, 5000 cells per well in 96-well plates were cultured for 48 hr and pulsed with 13H]thymidine for 4 hr, and specifically incorporated radioactivity was measured. Purification of SCF from ERG3A-Conditioned Medium BRL9A cells (Coon, 1968; obtained from ATCC) were grown in a 20 liter fermenter (Biolafitte) perfusion culture system. Cells were inoculated at a density of 3 x lo9 cells per ml onto 5 g/liter Cytodex 2 microcarriers (Pharmacia) in minimal essential medium (with Earle’s salts) (GIBCO), 2 mM glutamine. 3 g/liter glucose, 2.95 g/liter tryptose phosphate, 5% FBS, and 5% fetal calf serum. The cells were allowed to attach and grow for 8 days, with perfusion of fresh medium maintaining glucose at a concentration of 1.5 g/liter. Serum-free medium was then perfused through the reactor, and conditioned medium was collected, concentrated by ultrafiltration, and diafiltered at 4OC against 50 mM Tris-HCI (pH 7.8) in batches ranging from 27 liters to 161 liters. Total conditioned medium used forthe purification described in Figure 2 and Table 1 was 336 liters. All subsequent steps were performed at 4OC. Batches were applied to DEAE-cellulose anion exchange columns (Whatman DE-52) equilibrated in 50 mM Tris-HCI (pH 7.8) (column sizes were about 100 ml bed volume per 10 liters of original conditioned medium). The columns were washed with start buffer and eluted with gradients (lOcolumn volumes)from 0 to 300 mM NaCl in theTris buffer. For the chromatography run illustrated in Figure 2A, sample was derived from a 41 liter batch of medium, column size was 5 x 20.4 cm, wash volume after sample application was 2 liters, total gradient volume was 4 liters, flow rate was 167 mllhr, and size of fractions (collected during application of the NaCl gradient) was 15 ml. Fractionswith HPPCFC activity were pooled. Pools from all of the DEAE-cellulose columns were combined, concentrated to 74 ml, and applied to an Ultrogel AcA 54 gel filtration column (LKB; 5 x 143.5 cm) equilibrated in 50 mM Tris-HCI, 50 mM NaCl (pH 7.4). Fractions of 14 ml were collected and flow rate was 70 mllhr. Fractions containing HPP-CFC activity were pooled and applied in two separate chromatographic runs to a wheat germ agglutinin-agarose column (5 x 24.5 cm; resin from E-Y Laboratories, San Mateo, CA) equilibrated in 20 mM Tris-HCI, 500 mM NaCl (pH 7.4). After washing with about 220 ml of column buffer, bound material, which included material active in the HPP-CFC and MC19 assays, was eluted by applying a solution of 350 mM N-acetyl-oglucosamine dissolved in the column buffer. Fractions of 13.25 ml were

Cell 200

collected at a flow rate of 122 mllhr. The active material from the wheat germ agglutinin step was dialyzed against 25 mM sodium formate (pH 4.2) centrifuged to remove the precipitate that appeared during dialysis and applied to an S-Sepharose Fast Flow (Pharmacia) cation exchange column (3.3 x 10.25 cm) equilibrated in the sodium formate buffer. Flow rate was 465 mllhr and fractions of 14.2 ml were collected. After sample application, the column was washed with 240 ml of column buffer and elution of bound material was carried out by applying a gradient of O-750 mM NaCl (NaCI dissolved in column buffer; total gradient volume 2200 ml). Pooled fractions from the S-Sepharose column were combined with an equal volume of buffer B (100 mM ammonium acetate [pH 6.0]:isopropanol, 25:75) and applied to a C4 reverse-phase column (Vydac Proteins Cq; 2.4 x 2 cm) equilibrated with buffer A (60 mM ammonium acetate [pH 6,0]:isopropanol, 62.537.5). After washing the column with 200 ml of buffer A, a linear gradient from buffer A to buffer B (total gradient volume 140 ml) was applied, and fractions of 9.1 ml were collected. Flow rate was 540 mllhr during sample application and 154 mllhr subsequently. Roughly 75% of the recovered activity ran through the first Cq column. This material was rechromatographed using C4 resin essentially as for the first run except that column size was 1.4 x 7.8 cm and flow rate was 50-60 mllhr throughout; roughly 50% of the activity emerged in the runthrough in this case, and 50% in the gradient fractions in purified form. Glycosidase lbatments Conditions for the glycosidase treatments of Figure 3 were 5 mM 3-((3cholamidopropyl]dimethylammonio)-l-propanesulfonate (CHAPS), 33 mM 2-mercaptoethanol, and 10 mM Tris-HCI (pH 7.0-7.2) for 3 hr at 37%. Neuraminidase (from Arthrobacter ureafaciens; Calbiochem) was used at 0.23 U/ml final concentration. O-glycanase (Genzyme; endo-a-N-acetyl-galactosaminidase) was used at 45 milliunits/ml. N-glycanase (Genzyme; peptide:N-glycosidase F; peptide-N4-[N-acetyl-l3-glucosaminyl]asparagine amidase) was used at 10 U/ml. N-Terminal Amino Acid Sequence Analysis of SCF Sequence analysis was performed with an automated amino acid sequencer, model 477 (Applied Biosystems, Inc., Foster City, CA) equipped with an online phenylthiohydantoin-amino acid analyzer and model 900 data analysis system (Hunkapiller et al., 1986). SDSPAGE followed by electroblotting onto PVDF membrane (Immobilon I? Millipore Corp.) was performed according to Matsudaira (1967). PVDF membrane pieces containing the blotted SCF bands were then excised and subjected to sequencing. Treatment with calf liver pyroglutamate aminopeptidase (Sigma) was carried out as described by Podell and Abraham (1978). Other Methods SDS-PAGE was carried out according to ing gels contained 12% (w/v) acrylamide. done according to Morrissey (1961). lz51 done by the chloramineT method (Hunter and mlL-3 were recombinantly expressed pure, active form. Murine GM-CSF was

Laemmli (1970). The separatSilver-staining of gels was labeling of purified SCF was and Greenwood, 1982). hll-6 in E. coli and recovered in from Genzyme.

Acknowledgments We thank James A. Miller for his contributions toward the purification of SCF and Helen Hockman for iodination of SCF. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “sdvertisement” in accordance with 16 USC Section 1734 solely to indicate this fact. Received

August

15, 1990.

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on

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hematopoietic

colony

Coffman, R. L. (1982). Surface antigen expression and immunoglobulin gene rearrangement during mouse pm-6 cell development. Immunol. Rev. 69, 5-23. Coon, H. G. (1968). Clonal culture Biol. 39, 29a.

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