PROTEIN

EXPRESSION

AND

PURIFICATION

1,

54-62

(1990)

Purification and Partial Characterization of Recombinant Human Differentiation-Stimulating Factor Charles

H. Schmelzer,’

Louis E. Burton, and Cathleen M. Tamony

Department of Recovery Process Research and Development, South San Francisco, California 94080

Received

April

13, 1990,

and

in revised

form

June

Genentech,

San Bruno

Boulevard,

21, 1990

Recombinant human differentiation-stimulating factor (rhD-factor) has been isolated to >95% purity from Chinese hamster ovary cells. RhD-factor is a glycoprotein with an apparent molecular weight of 45.6 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On gel filtration in 6 M guanidinehydrochloride, rhD-factor elutes with an apparent molecular weight of 21.5 kDa; it elutes with an apparent molecular weight of 44.8 kDa under neutral pH (native) conditions. The amino-terminal sequence (12 residues) is consistent with the expected sequence derived from the genomic DNA sequence. Recombinant D-factor is heavily glycosylated with 30% by weight neutral sugar and 12% sialic acid. The ED,, for rhD-factor was 0.25 rig/ml. Trifluoromethanesulfonic acid-deglycosylated rhD-factor has a biological activity comparable to that of the native recombinant protein (ED,, = 0.40 rig/ml). The biological activity of rhD-factor was stable at pH 1 for 40 h, in 6 M guanidine-HCl containing buffers with or without reducing agent, and in 1% SDS. Carboxymethylation of D-factor after reduction totally destroyed biological activity. o 1990 Academic PESS, IUC.

A number of factors that affect the proliferation and differentiation of hematopoietic stem cells have been described (1,2). Several distinct colony-stimulating factors (CSFs)’ have been shown to cause the differentia’ To whom correspondence should be addressed. * Abbreviations used: rhD-factor, recombinant human differentiation-stimulating factor, hereafter referred to as D-factor, unless otherwise indicated; MGI-2, macrophage and granulocyte inducer 2; Mops, 3-(N-morpholinolpropanesulfonic acid; PBS, phosphate-buffered saline; TFA, trifluoroacetic acid; AcN, acetonitrile; RP-HPLC, reversed-phase high-performance liquid chromatography; RCM, reduced and carboxymethylated; Gdn-HCl, guanidine-hydrochloride; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; LIF, leukemia inhibitory factor; CSF, colony-stimulating factor; M-CSF, macrophage CSF; G-CSF, granulocyte CSF, GM-CSF, gran54

Inc., 460 Point

tion and proliferation of normal bone marrow granulocyte or macrophage precursor cells (3,4) including GMCSF, IL-3, G-CSF, and M-CSF. In studies of regulation by differentiation factors, a number of investigators have focused on the murine myeloid leukemic cell line Ml. Myeloid leukemic cells appear to be uncoupled with respect to differentiation and proliferation whereas normal blood stem cells are closely regulated, balancing the two functions (4). Treatment of Ml cells with a variety of factors has resulted either in induction of cell growth and differentiation (G-CSF) (5) or in their inhibition (IL-la, IFN-P, TNF-LY) (6). Combinations of agents such as IL-lo and IFN-P, or TNF-(w, will continue to inhibit growth while inducing phagocytosis (6,7). D-factor (8-lo), LIF (ll-13), and MGI-2 (14) are factors that have been shown to induce differentiative effects in Ml cells but not to promote colony formation (10,ll). These proteins are distinct from the CSFs and lymphokines (8,9), with current evidence suggesting that all three are the same factor (8,11,14-17). Natural D-factor has been purified and partially characterized from mouse L929 cells (8,18), rat YS cells (19), mouse spleen lymphocytes (9), and mouse Erhlich ascites cells (10). LIF has been purified, characterized (11,12), and partially cloned (11) from Krebs II cells. Recently, a cholinergic neuronal differentiation factor has been purified from cultured rat heart cells and has been shown to be identical to D-factor (20). Human D-factor has recently been cloned and expressed (21), showing that the factor from Ehrlich cells and LIF from Krebs II cells are identical. We report here a rapid, simple purification procedure for recombinant human D-factor expressed in CHO ulocyte and macrophage CSF, TNF, tumor necrosis factor; IL, interleukin; IFN, interferon; CHO, Chinese hamster ovary; SSFF, S-Sepharose Fast Flow; TFMS, trifluoromethanesulfonic acid; WGA, wheat germ agglutinin; Con A, concanavalin A; GlcNac, N-acetylglucosamine; BSA, bovine serum albumin, TCF, tissue culture fluid. 1046-5928/90 $3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

PURIFICATION

AND

CHARACTERIZATION

cells, which yields substantial amounts of D-factor enabling us to more thoroughly investigate the structural and functional aspects of this protein. EXPERIMENTAL

PROCEDURES

Materials S-Sepharose Fast Flow, Sephacryl S-300, low-molecular-weight standards, isoelectric focusing standards (310), PhastGel IEF 3-9, polybuffer 96, polybuffer 74, a PD-10 (Sephadex G-25 M) column, a Mono P HR5/20 column, and a Superose 12 HR lo/30 column were purchased from Pharmacia LKB Biotechnology, Inc. YMlO ultrafiltration membranes and a stirred cell concentrator were from Amicon. A microdialysis system was purchased from Bethesda Research Laboratories. Bio-Lyte 3110 carrier ampholytes were from Bio-Rad. Anisole (29,629-5), TFMS (15,853-4), and ether (17,926-4) were from Aldrich. Iodoacetic acid (1371) was from Kodak. Clostridium perfringens neuraminidase type X (N2133), myoglobin (M9267), P-lactoglobulin A (L5137), trypsin inhibitor (T1021), carbonic anhydrase (C6403), ovalbumin (A2512), cytochrome c (C7752), BSA, aprotinin (A1153), blue dextran (D4772), 2-mercaptoethanol, dithiothreitol, dichloromethane (17-3), and pyridine (P4036) were purchased from Sigma. All other chemicals were of reagent grade. Methods Purification of D-factor. All chromatographic steps were performed at room temperature unless otherwise indicated. D-factor was transfected into CHO cells (Lowe and Shepard, unpublished experiments) using the vector described in Ref. (21). Tissue culture supernatants containing D-factor were provided by the Genentech cell culture group. Tissue culture fluid (TCF, 3.2 liters) containing D-factor from CHO cells was concentrated at 4°C approximately &fold using an Amicon stirred cell concentrator (YMlO membrane). The concentrated TCF was dialyzed overnight with 2 X 18 liters of 25 mM sodium succinate, pH 4.0. The dialyzed supernatant was applied to a 2.5 X lo-cm SSFF column equilibrated in 25 mM succinate, pH 4.0, at a flow rate of 5 ml/min. After the sample was loaded, the column was washed with equilibration buffer until the A, was near baseline. The column was eluted with a O-O.4 M NaCl gradient in 25 mM succinate, pH 4.0. Fractions containing D-factor (as determined by SDS-PAGE) were pooled, concentrated approximately &fold, and diafiltered into 25 mM succinate, pH 4.0, 0.1 M NaCl in a stirred cell using a YMlO membrane. The concentrated pool was applied to a Sephacryl S-300 HR column (5 X 89 cm) equilibrated with 25 mM succinate, pH 4.0,O.l M NaCl at a flow rate of 3 ml/min at 4°C. Fractions containing D-factor were pooled, concentrated 3.5-fold,

OF

D-FACTOR

and stored at 4“C for biological izations.

55 and chemical character-

SDS-polyacrylamide gel electrophoresis. Slab gel electrophoresis was performed in a 12.5% resolving gel with a 4% stacking gel (22). The gels were silver stained for protein detection (23). Isoelectric focusing. The pI for D-factor was determined using a Pharmacia Phast gel system (PhastGel IEF 3-9). Prior to use, the IEF gel was soaked in BioLyte 3110 carrier ampholyte solution (1:lO) containing8 M deionized urea and 0.025% Triton X-100. The pH gradient was determined using calibrated pl standards (broad pl calibration kit 3-10): myoglobin (6.76,7.16), glucose oxidase (4.2), carbonic anhydrase (5.85), trypsin inhibitor (4.55), and P-lactoglobulin A (5.13). Protein sequencing. The amino-terminal sequence of D-factor was determined using an Applied Biosysterns 477 sequenator with an on-line 120A HPLC. Amino acid analysis. Amino acid compositions were determined using a Beckman 6300 amino acid analyzer. Samples were hydrolyzed for 24 h at 1lO’C in 6 N HCl prior to analysis. Amino sugar analysis. Protein samples were hydrolyzed for 2 h at 110°C. Glucosamine was quantitated on a Beckman 6300 amino acid analyzer using a sulfonated polypropylene Na-high performance column at 84°C. Gel filtration. The molecular weight of D-factor was determined in denaturing media by gel filtration on a Superose 12 column (1 X 30 cm) equilibrated in 25 mM Tris-HCl, pH 7.2, containing 6 M guanidine-hydrochloride (flow rate, 0.5 mUmin). Reduced carboxymethylated D-factor samples (-30 pg) were prepared prior to chromatography by reduction at 37°C for 4 h followed by 90°C for 10 min in 0.5 M Tris-HCl, pH 8.5, containing 6 M guanidine-hydrochloride and 500- to lOOO-fold molar excesses of 2-mercaptoethanol over disulfide bonds. Reduced D-factor was then alkylated with a lofold molar excess of iodoacetic acid over total sulfhydryl groups. The reagents were removed either by dialysis or by gel filtration into column equilibration buffer. Other samples were dissolved in equilibration buffer at room temperature prior to chromatography. The Superose 12 column was calibrated using the following nonreduced standards: bovine serum albumin (67,000), ovalbumin (43,000), carbonic anhydrase (30,000), myoglobin (17,200), and cytochrome c (12,400). The molecular weight and Stokes radius of D-factor were determined by gel filtration on a Superose 12 column equilibrated in 50 mM Mops, pH 7.0, containing 0.5 M NaCl. Stokes radii were calculated using the equation of Laurent and Killander (24). Chromatographic conditions were the same as those above except for the substitution of Mops/NaCl buffer for guanidine hydrochloride. The Superose 12 column was calibrated using the

56

SCHMELZER,

BURTON,

AND

TAMONY 1234

I

jc&

1

67.0

-

43.0

-

30.0

-

2

3

4

5

*

J- - - -,- - - - ---/J9 I li 200 , 0

400

I

600

,

1000

600

Volume

0

(ml)

FIG. 1. S-Sepharose Fast Flow chromatography. The concentrated CHO tissue culture media were dialyzed and then applied to a 2.5 X lo-cm column equilibrated in 25 mM succinate, pH 4.0. The column was eluted with a O-O.4 M NaCl gradient in 25 mM succinate, pH 4.0. Absorbance at 280 nm was monitored continuously. Fractions containing D-factor were pooled as indicated by brackets.

nonreduced standards, bovine serum albumin, ovalbumin, carbonic anhydrase, myoglobin, and cytochrome c. Chromatofocusing. Chromatofocusing was performed on a Mono P HR 5/20 column. The column was equilibrated in 25 mM diethanolamine, pH 9.2, containing 4 M urea. The flow rate was 0.5 ml/min and 1.25min fractions were collected. The absorbance of the column

1

i

I

0.4 -

0.3 F z cx $

0.2 -

j $

0.1 -

0’

I

I

I

I

BOO

1000

1200

1400

Volume (ml) FIG. 2. Elution profile of protein from the Sepharose S-300 column. The active fractions from the S-Sepharose Fast Flow column were applied to a 5 X 89-cm column equilibrated in 25 mM succinate, pH 4.0, 0.1 M NaCl. Fractions were analyzed by SDS-PAGE. Fractions containing D-factor were pooled as indicated by brackets.

FIG. 3. SDS-polyacrylamide gel electrophoresis of D-factor. (A) Fractions at each stage of purification: lane 1, molecular weight markers; lane 2, dialyzed tissue culture media supernatant; lane 3, S-Sepharose fast flow pool; lane 4, Sephacryl S-300 pool. (B) Lane 1, purified D-factor (nonreduced); lane 2, deglycosylated D-factor (nonreduced); lane 3, molecular weight markers; lane 4, purified Dfactor (reduced); lane 5, deglycosylated D-factor (reduced).

effluent was monitored continuously at 280 nm. Selected fractions were also analyzed by SDS-PAGE. Samples were prepared for chromatofocusing by desalting into buffer A (25 mM diethanolamine, pH 9.2, containing 4 M urea) using a PD-10 column. Each sample of D-factor was then injected onto the Mono P column and eluted with buffer A. Once the absorbance returned to baseline, the column was eluted with the following buffers: buffer B, 10% polybuffer 96/4 M urea, pH 6.0; buffer C, 10% polybuffer 7414 M urea, pH 4.0; and buffer D, 25 mM succinate/l M NaC1/4 M urea, pH 4.0. Bioassay. D-factor activity was measured by the inhibition of [3H]thymidine uptake by Ml-T22 cells (21). Cells (1 X 105/well) were incubated with varying amounts of D-factor for 72 h at 37”C, pulsing for 4 h with 1 mCi of [3H]thymidine. ED,, values were calculated using a four-parameter curve-fitting program (25). D-factor was Stability studies. For SDS treatment M NaCl, dialyzed overnight into 50 mM Tris-HCU0.1 pH 7.5, and then SDS was added to a final concentration of 1%. After approximately 1 h of incubation with SDS at room temperature, D-factor was diluted into culture media. For reduction and carboxymethylation treatment, D-factor was dialyzed into 0.5 M Tris-HCl/G M guanidine hydrochloride, pH 8.5. A control sample was removed and dialyzed into 50 mM Tris-HCU0.1 M NaCl, pH 7.5. A second aliquot was reduced with a lofold molar excess of dithiothreitol over disulfide bonds for 6 h at 37°C followed by dialysis into 50 mM TrisHCU0.1 M NaCl, pH 7.5. A third aliquot was reduced as indicated above and then carboxymethylated with a 5fold molar excess of iodoacetic acid over total sulfhydryl groups for 20 min at room temperature, followed by dialysis into 50 mM Tris-HCU0.1 M NaCl, pH 7.5. All sam-

PURIFICATION

AND

CHARACTERIZATION TABLE

OF

57

D-FACTOR

1

Purification of Recombinant Human D-Factor from CHO Cells

Step Tissue culture (dialyzed) SSFF s-300

fluid

Volume (ml)

Protein bd

430 165 45

215 58 53

Total

units

Specific activity V-Jhd

Purification (fold)

Recovery (%I

ED, (n&-4

concn 17.4 46.2 31.9

ples were then diluted into culture media and assayed for biological activity. Protein assays. Protein concentrations were determined by the method of Bradford (26) using the Pierce protein assay reagent. D-factor quantitated by amino acid analysis was used as the standard. Protein concentrations of purified samples were also determined by amino acid analysis or by absorbance at 280 nm using experimentally determined t”.lX* ’ Cm= 0.67. Neuraminidase treatment. D-factor (1 mg) was incubated with 1 unit of C. perfringens neuraminidase for 15 h at 37°C in 50 mM sodium acetate, pH 5.1. Deglycosylation. D-factor was deglycosylated by treatment with trifluoromethanesulfonic acid as previously described (27). The following modifications were made: 2.0 mg of D-factor was vacuum-dried both prior to and after the methylene chloride wash; the dried sample was suspended in 20 ~1 anisole and 180 ~1 TFMS; the incubated TFMS reaction mixture was

TABLE

0.36 0.8 0.6

Theoretical“

Asx Thr Ser Glx Pro GUY Ala CYS Val Met Be Leu TY~ Phe His LYS Arg

21 10 10 12 11 12 14 6 11 1 10 25 7 6 6 12 6

0.39 0.16 0.20

2 TABLE

Observed* 20.8 9.7 8.8 11.7 10.6 12.7 13.7 2.5 11.0 0.2 11.2 26.9 7.7 6.0 5.9 12.2 5.8

f f + * k r+ f + k + + + + k k k 2~

sequence. of D-factor,

0.6 0.3 0.4 0.8 0.7 0.6 0.2 0.2’ 0.1 0.1 1.4 0.9 0.4 0.2 0.2 0.5 0.6

Cycle

+ SD.

Residue

1 2 3 4 5 6 7 8 9 10 11 12

the mean

3

Amino-Terminal Sequence Analysis of Recombinant D-Factor

D No amino a Based on the human gene b Based on nine preparations ’ As cysteine.

100 60 41

added to 4 ml of pyridine/diethyl ether (l/9); and the suspended pellet was dialyzed against 0.05 M acetic acid and prepared for SDS-PAGE. Neutral sugar analysis. Neutral sugars were quantitated using the phenol sulfuric acid method (28). DMannose was used as the standard and the absorbance was read at 492 nm. Sialic acid determination. Sialic acid was determined using the dimethoxydiaminobenzene method (29). Lectin affinity chromatography. WGA-Sepharose: Separately, either 200 pg of native D-factor or 100 pg of deglycosylated D factor was applied to a l-ml WGASepharose column, washed with 1X PBS, and eluted with 5 column volumes of 0.5 M N-acetyl-D-glucosamine. Column fractions were dialyzed into 0.1 M acetic acid, dried, and analyzed by SDS-PAGE. Con A-Sepharose chromatography: Separately, 100 pg of native Dfactor or 200 pg of deglycosylated D-factor was loaded onto a l-ml Con A column, previously equilibrated in PBS, washed with PBS, and eluted with 5 column volumes of 0.5 M cr-methyl-D-glucopyranoside in PBS.

Amino Acid Analysis of Recombinant Human D-Factor Residue

1 2.2 1.7

Ser Pro Leu Pro Ile Thr Pro Val Asn” Ala Thr cks*

Amount bmol) 315 304 310 276 251 217 170 218 245 182

acid was found in this cycle, but Asn is consistent probably is glycosylated. acid was found in this cycle, but Cys is expected

with

sequenceand

’ No amino the sequence.

from

58

SCHMELZER, TABLE

BURTON,

AND

TAMONY

4

PH

123

Molecular Weight Determination of D-Factor by Size-Exclusion Chromatography

Sample D-Factor Native RCM Deglycosylated Native RCM

M, (Gdn-HCl)

Stokes radius” 6)

M, (Mops/NaCl)

32,500

44,800 49,900

28.7 30.3

14,100

23,400

24.0

22,800

N.D.*

N.D.

21,500

5.85 -

D-factor

7.18

’ Determined in 50 mM Mops, ’ Not determined.

pH

7.0, containing

0.5 M NaCl.

FIG. = 4.2);

5.

Isoelectric focusing lane 2, D-factor; lane

of D-factor. 3, carbonic

Lane 1, glucose oxidase anhydrase (pZ = 5.85).

(pZ

terminal sequence analysis, Fig. 3A, lane 4, and Table 3) from 3.2 liters of CHO tissue culture fluid (Table 1).

RESULTS Purification We describe here a two-step chromatographic purification of D-factor from tissue culture fluid. We concentrated approximately 3.2 liters of tissue culture fluid in a stirred cell to approximately 460 ml and dialyzed into 25 mM succinate, pH 4.0. The supernatant was chromatographed on a SSFF column as shown in Fig. 1. The fractions containing D-factor were pooled, concentrated approximately B-fold, and diafiltered into 25 mM succiSSFF pool nate, pH 4.0, 0.1 M NaCl. The concentrated was chromatographed on Sephacryl S-300 HR in 25 InM succinate, pH 4.0, 0.1 M NaCl (Fig. 2). Fractions containing D-factor (determined by SDS-PAGE) were pooled, concentrated 3.5-fold, and subsequently used for protein characterization. As can be seen in Fig. 3A, lane 3, there is substantial purification of D-factor by the SSFF column. We recovered 53 mg of D-factor (>95% purity as determined by SDS-PAGE and amino-

nglml

D-Factor

FIG. 4. Biological potency of D-factor. Biological assay conditions are described under Experimental Procedures. D-factor, open circles; deglycosylated D-factor, filled circles.

Characterization

of Native rhD-Factor

The amino acid composition of D-factor is consistent with the theoretical composition expected from the genomic sequence (Table 2) (21). The amino-terminal sequence analysis (12 residues) of the purified D-factor pool (Table 3) was consistent with the expected amino terminus deduced from the DNA sequence. The molecular weight of D-factor determined by SDS-PAGE was 45,600 + 800 Da, and it was 46,600 + 800 Da following reduction (data not shown). The molecular weights of D-factor determined by gel filtration were 21,500 Da in guanidine-HCl and 44,800 Da in Mops/NaCl (Table 4). The molecular weight of D-factor is 19,700 Da on the basis of the DNA sequence. After reduction and carboxymethylation, the molecular weight of D-factor determined by gel filtration increases to 32,500 Da in guanidine-HCl, and reduced carboxymethylated D-factor was 49,900 Da in Mops/NaCl (Table 4), indicating the possibility that intramolecular disulfide bonds are present in the molecule. The Stokes radii for D-factor were 28.7 A before and 30.3 A after reduction (Table 4). D-factor had an ED,, of 0.25 + 0.12 rig/ml (n = 7) as determined in the Ml-22 assay system (21) (Fig. 4). The isoelectric focusing pattern of D-factor under nonreducing conditions revealed at least seven bands focusing between pH 4 and pH 5.85 (Fig. 5). Chromatofocusing results using purified D-factor revealed that there were a variety of different D-factor species and that some of these molecules did not bind to the Mono P column at pH 9.0, while other molecules were observed to bind and elute in the pH range 9 to 4 (Figs. 6A and 7). There were even some species of D-factor that evidently required 1 M NaCl to be eluted from the column (Figs. 6A and 7). Upon treatment of D-factor with neuraminidase the majority of the molecules were observed in the

PURIFICATION

AND

CHARACTERIZATION

OF

59

D-FACTOR

lb0 Fraction number

Fraction number

50 Fraction number

FIG. 6. Chromatofocusing of D-factor on Mono P column. D-factor samples were subjected to chromatofocusing on a column was equilibrated in buffer A (25 mM diethanolamine-HC1/4 M urea, pH 9.2) and bound protein was eluted using the following buffers: buffer B, 10% polybuffer 96-HC1/4 M urea, pH 6.0; buffer C, 10% polybuffer 74-HC1/4 M urea, pH mM succinate/4 M urea/l M NaCl, pH 4.0. (A) Elution profile of D-factor. (B) Elution profile of desialylated D-factor. deglycosylated D-factor.

flowthrough fractions (Figs. 6B and 7). The molecules that flowed through the column were probably devoid of sialic acid residues following neuraminidase treatment. The pl of D-factor determined by computer calculation3 (30) is 10.0, assuming that all the cysteinyl sulfhydryl groups are involved with intramolecular disulfide bonds. The glucosamine content was determined to be 19.5 f 1.2 mol/mol of D-factor by amino sugar analysis (Table 5). No galactosamine could be detected, indicating that D-factor probably does not contain any significant O-linked carbohydrate (data not shown). The neutral sugar content was determined to be 0.30 mg/mg D-factor or 30% neutral sugar. Sialic acid content was 0.12 mg/mg of D-factor or 12% (Table 5). Lectin affinity chromatography revealed that greater than 80% of the ’ The computer Richard Vandlen, pletely denatured ionizable groups.

program for pl calculation was written Genentech, Inc. The program assumes protein in which there is no interaction

by Dr. a combetween

Mono P column. The a sequential series of 4.0; and buffer D, 25 (C) Elution profile of

D-factor pool bound to both Con A-Sepharose and WGA-Sepharose and could be eluted with the appropriate specific sugar: a-methyl-D-glucopyranoside for Con A and N-acetyl-D-glucosamine for WGA (data not shown). The biological activity of D-factor was stable to exposure to pH 1 for 40 hs, to 6 M guanidine-HCl containing buffers with or without reducing agent, and to 1% SDS (Table 6). Incubation of D-factor with pepsin at pH 1 for 40 h totally destroyed biological activity, and a number of peptides were observed on HPLC (Table 6 and data not shown). Carboxymethylation of D-factor after reduction totally destroyed biological activity (Table 6). Characterization

of Deglycosylated

D-Factor

D-factor was deglycosylated by trifluoromethanesulfonic acid. Amino-terminal sequence analysis revealed only one sequence, that of native D-factor, indicating that no peptide bond cleavages had occurred during the deglycosylation reaction (data not shown). The amino

60

SCHMELZER.

1234567

BURTON.

AND

TAMONY TABLE

kDa Stability

6

of D-Factor Activity (% control”)

Treatment 1% SDS 6 M Gdn-HCl 6 M Gdn-HCl + DTT RCM pH 1 (40 h) pH 1 + pepsin* TFA/AcN ’ FIG. 7. SDS-polyacrylamide gel electrophoresis of D-factor chromatofocusing pools. Lane 1, native D-factor (2 pg); lane 2, flowthrough fraction from Fig. 6A; lane 3, D-factor that eluted from the Mono P column between pH 9 and 6 by buffer B (Fig. 6A); lane 4, D-factor that eluted from the Mono P column between pH 6 and 4 by buffer C (Fig. 6A); lane 5, buffer D eluting fraction from the Mono P column (Fig. 6A); lane 6, flowthrough fraction from desialylated Dfactor from Mono P column (Fig. 6B); lane 7, molecular weight markers.

acid composition of deglycosylated protein was consistent with the composition of native D-factor. The ED,, for deglycosylated D-factor was -0.4 rig/ml, suggesting that carbohydrate does not play a major role in biological activity (Fig. 4). The molecular weight of deglycosylated D-factor determined by SDS-PAGE was 20,100 Da and it was 21,400 Da following reduction (Fig. 3B). The molecular weights of deglycosylated D-factor determined by gel filtration were 14,100 Da in guanidine-HCl and 23,400 Da in Mops/NaCl (Table 4). After reduction, the molecular weight of the deglycosylated molecule determined by gel filtration increased to 22,800 Da in guanidineHCl (data not shown). The Stokes radius of deglycosylated D-factor determined by gel filtration in0 50 mM Mops, pH 7.0, containing 0.5 M NaCl, was 24.0 A (Table 4). The molecular weight and Stokes radius of reduced deglycosylated D-factor could not be determined in Mops/NaCl due to interactions with the Superose 12 resin (data not shown). Chromatofocusing of deglycosylated D-factor showed that most of the protein was in

115 100 86

Purification and partial characterization of recombinant human differentiation-stimulating factor.

Recombinant human differentiation-stimulating factor (rhD-factor) has been isolated to greater than 95% purity from Chinese hamster ovary cells. RhD-f...
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