- Email: [email protected]
[79] Induction and partial purification of duck fibroblast interferon
[79]
DUCK FIBROBLAST INTERFERON
2o3
different molecular species by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis is not feasible because type II interferon antiviral activity is irreversibly inactivated in the presence of SDS (unpublished observation). Antigenic Properties. As reported earlier, le'18 type II interferon cannot be neutralized by antisera directed against virus-induced interferon. The relatively large amounts of type II interferon prepared by the method described in this chapter made it possible to prepare a rabbit anti-type II mouse interferon serum. 11 This antiserum neutralized several T mitogeninduced interferons as well as the type II interferon that is induced in BCG-sensitized mice by injection of the specific antigen, tuberculin. Furthermore, the anti-type II interferon serum did not neutralize any of the type I interferons tested (virus-induced, lipopolysaccharide-induced, etc.). These results indicate that mitogen- and antigen-induced type II interferons are antigenically related, and confirm earlier observations about the different antigenic properties of type I and type II interferons. is j. Wietzerbin, R. Falcoff, L. Catinot, and E. Falcoff, Ann. lmmunol. (Paris) 128 C, 699 (1977).
[79] I n d u c t i o n
and Partial Purification of Duck Fibroblast Interferon
By ROBERT E. ZIEGLER and
WOLFGANG
K.
JOKLIK
Duck interferon is a useful reagent for studying the multiplication of RNA tumor viruses. Duck cells form a very useful system for this purpose for two reasons: they contain very little endogenous RNA tumor virus genetic information, certainly less than one complete genome; and certain cleavages of precursor to product proteins occur more slowly in duck embryo fibroblasts than in other cells. One example in this connection is the fact that RNA-dependent DNA polymerase can be isolated from RNA tumor viruses grown in duck embryo fibroblasts (DEF) not only in the a/3 and the a forms, but also in a/32 form, which is the precursor of the other two1; by contrast, very little if any of the/32 form can be isolated from RNA tumor viruses grown in chick embryo fibroblasts (CEF). Further, DEF form monolayers that are in many respects easier to handle and more uniform than CEF. It should be noted that chick interferon fails to protect DEF against A. Hizi and W. K. Joklik, J. Biol. Chem. 252, 2281 (1977).
METHODS IN ENZYMOLOGY, VOL. 78
Copyright © 1981by AcademicPress, Inc. All fights of reproduction in any form reserved. ISBN 0-12-18197g-7
564
PURIFICATION AND CHARACTERIZATION
[79]
viral infection; the titer of chick interferon on DEF is less than one-tenth the titer on CEF. Composition of Media, Buffers, and Solutions GM: Ham's F10, 10% (v/v) tryptose phosphate broth, 50 units of penicillin per milliliter, 50 ~g of streptomycin per milliliter, 2/zg of fungizone per milliliter, 5% calf serum (pH 7.2). PGM: GM plus 3% extra calf serum (total calf serum concentration equals 8%) and 2% chick serum (pH 7.2) LSMG: GM with only 0.1% (v/v) calf serum instead of 5% OLMC: 2x GM, 50/zg of gentamycin per milliliter PBSI: 0.14 M NaC1, 8 mM Na~HPO4, 1.5 mM KH2PO4, 2.5 mM KC1 (pH 7.0) Puck's saline A: 0.8% NaC1, 0.04% KCI, 0.009% Na~HPO4.H~O, 0.006% KHzPO4, 0.1% glucose, 0.035% NaHCOs, 0.4% MgClz.6 H~O, 0.002% phenol red (pH 7.2) Buffer L: 150 mM NaC1, 50 mM sodium phosphate (pH 7.2) Buffer Wl: Same as buffer L Buffer W2:0.40 M NaC1, 50 mM sodium phosphate (pH 7.2) Buffer W3:0.60 M NaC1, 50 mM sodium phosphate (pH 6.5) Buffer W4:0.80 M NaC1, 50 mM NaCOOCH3 (pH 5.5) Buffer El: 0.80 M NaCI, 50 mM NaCOOCHs (pH 4.5) Buffer E2:0.50 M NaCI, 50 mM EDTA Preparation of Duck E m b r y o Fibroblasts Primary DEF are prepared from 12- to 13-day-old Peking duck embryos by a modification of the method of Vogt. 2 Briefly, after removing, under sterile conditions, the shell and outer membrane, embryos are removed from the eggs with a sterile rectal hook and are placed in a 150-mm plastic tissue culture dish containing sterile PBS1. The embryos are decapitated and eviscerated using sterile forceps, rinsed in a second tissue culture dish containing PBS1, and placed into a third dish containing fresh PBS1. This is repeated for 15-20 embryos per preparation. A 0.25% (v/v) trypsin solution, 10 ml per embryo, is added to a 300-ml trypsinizing flask containing a magnetic stirring bar. The embryos are placed into a sterile 10-ml glass syringe fitted with a 1.5-inch, 16-gauge needle and are then passed through the needle into the trypsinizing flask. Ten embryos are trypsinized in each 300-ml flask. 2 p. K. Vogt, in "Fundamental Techniques in Virology " (K. Habel and N. P. Salzman, eds.), p. 198. Academic Press, New York, 1969.
[79]
D U C K FIBROBLAST I N T E R F E R O N
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The trypsin-embryo suspension is stirred for 15 min at 37°, the flask is tilted, the larger debris are allowed to settle, and the supernatant containing the trypsinized cells is poured sterilely into centrifuge bottles containing 20 ml of cold PGM per embryo. If the extent of digestion of the embryos is insufficient, the trypsinization steps are repeated. The cells are pelleted (1500 rpm, 15 min, 4°), the supernatant is discarded, and the cell pellet is resuspended in warm PGM (10 ml per embryo). The cells are counted, after diluting 0.2 ml of the cell suspension into 10 ml of PBS1, with a Coulter counter Model FN with an attenuation setting of 1, an aperture setting of 128, and a threshold setting of 25. The cells are seeded into 150-mm plastic tissue culture dishes in 25 ml of PGM (1.2 × 107 cells/dish) or into 1330-cm~ glass roller culture bottles in 200 ml of PGM (2.5 × 108 cells/roller bottle). Tissue culture dishes are incubated at 37° in a water-jacketed incubator in a 5% CO2/95% air atmosphere. Roller culture bottles are placed onto a Bellco roller bottle apparatus in a 37° warm room and are rotated at 6 revolutions/hr. Monolayers are confluent after 48 hr. Assay of Duck Interferon Duck interferon is assayed either by a microtiter cytopathic effect (CPE) reduction or by a plaque reduction assay, in both cases with vesicular stomatitis virus (VSV) grown in DEF. It should be noted that VSV grown in CEF is essentially incapable of infecting DEF. Microtiter CPE Reduction Assay. Duck interferon is assayed by a CPE reduction assay combining the techniques of Henderson and Joklik 3 and Tilles and Finland. 4 Briefly, 100/zl of GM are added to each well of a Linbro microtiter 96-weU tissue culture plate (8 rows of 12 wells each) with an 8 × 100 Izl Titertek manifold. One hundred microliters of each duck interferon sample to be assayed is added to the first well of each row, and simultaneous serial twofold dilutions are performed with an 8 x 10/xl manifold, a 100-/zl row being discarded at the end of the last dilution (final volume, 100/zl/well). To each well 4 × 104 DEF are added in 50/zl of GM with an 8 × 50/zl manifold, and the plates are incubated for 24 hr at 37°, during which time the cells attach to the surface of the well to form monolayers and, if interferon is present, become resistant to virus challenge. The monolayers are challenged with 200 plaque-forming units (PFU) of VSV per well in 50/zl of GM at the end of the 24-hr incubation period. The plates are incubated for an additional 36 hr at 37°, and the monolayers are then examined under a light microscope for evidence of 3 D. R. H e n d e r s o n and W. K. Joklik, Virology 91, 389 (1978). 4 j. G. Tiiles and M. Finland, Appl. Microbiol. 16, 1706 (1968).
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PURIFICATION AND CHARACTERIZATION
[79]
virus-induced CPE. Control monolayers with no duck interferon added show complete destruction of cell monolayers. In interferon-treated cell monolayers, the change from complete protection to complete destruction of the cell monolayer usually occurs within a range of three consecutive wells. The titer of an interferon sample in microtiter units (MTU) is defined as the reciprocal of that dilution which is halfway between the highest dilution of interferon that completely protects the cell monolayer and the lowest dilution of interferon that allows the complete destruction of the cell monolayer. Plaque Reduction Assay. The assay used is that of Lai and Joklik. 5 D E F are seeded into 60-mm tissue culture dishes at a density of 2 x 106 cells/dish. When the monolayers are confluent, the medium is removed from the dishes, and 3-ml aliquots of serial dilutions in GM of interferon preparations are added to duplicate dishes. The dishes are incubated for 24 hr at 37°, the interferon-containing medium or control medium is removed, 100 PFU of VSV are added to each dish in 0.1 ml of GM, the dishes are incubated at 37° for I hr, and each dish is then overlaid with 5 ml of a mixture of 41% (v/v) prewarmed agar and 59% (v/v) OLMC. The overlay is allowed to harden at room temperature, and the dishes are incubated at 37° until plaques are visible on control plates by light microscopy (about 36 hr.) One milliliter of a neutral red solution is then added to each dish, the dishes are incubated for an additional 6 hr at 37°, and the plaques are counted using a light box with indirect illumination. One plaque-reduction unit of duck interferon (1 PRDs0) is defined as that dilution of a standard duck interferon preparation that reduces the number of plaques developing in an interferon-treated DEF monolayer to 50% of the number of plaques developing in the control monolayer. Under these conditions 1 MTU is equivalent to 2 PRDs0. Induction of Duck Interferon The optimum inducer of interferon in DEF is reovirus. DEF are nonpermissive for reovirus. The effect of several variables on the yield of duck interferon were studied; among them were (a) the effect of varying reovirus multiplicity; (b) the effect of irradiating reovirus with various doses of ultraviolet irradiation; (c) the effect of varying the length of the incubation period following the initial induction period; (d) the effect of varying the concentration of serum in the incubation medium; and (e) the effect of varying the incubation medium volume. As a result of these studies, the most efficient method of inducing the synthesis of interferon in confluent monolayers of DEF has been determined to be as follows. s M.-T. Lai and W. K. Joklik,
Virology 51, 191 (1973).
[79]
DUCK FIBROBLAST INTERFERON
567
Medium is removed from confluent monolayers of DEF (2.5 x l08 cells) growing in 1330-cm 2 glass roller culture bottles, and the cells are inoculated with 50,000 particles (about 250 PFU)/ceU of reovirus serotype 3 (strain Dearing) 6 suspended in 10 ml of a 1 : 1 mixture of Puck's saline A and LSGM. After 2 hr at 37°, 40 ml of LSGM are added, and the bottles are incubated for 58 hr at 37°, when the interferon-containing medium is harvested. This induction process yields about 2.5 x 104 MTU of interferon per 107 cells (about 6 x 105 MTU per bottle). Purification of Duck Interferon
General Considerations. The purification of duck interferon is divided into three steps: (a) an acidification and clarification step; (b) a concentration step; and (c) a column chromatography step. Each step is discussed below. Duck interferon has several properties that affect the manner in which it can be purified: (a) it is sensitive to dialysis, more so with increasing purity; (b) it is rapidly inactivated by exposure to sodium dodecyl sulfate (SDS) in concentrations of 0.01-1.0% (v/v) and to 0.01% (v/v) 2-mercaptoethanol (2-ME); and (c) it adsorbs readily to glass and must therefore be stored in polypropylene containers. Initial Processing of Crude Duck Interferon. The harvested medium containing crude interferon is centrifuged to remove cellular debris (2000 rpm, 20 min, 4°) and acidified to pH 2; it can then be stored at 4° for at least 2 months without loss of activity. The acidified, crude interferon preparation is then centrifuged at 40,000 rpm for 45 rain at 4° to remove residual reovirus, precipitated protein, and other debris. Concentration of Crude Duck Interferon. Several methods were tested for their ability to concentrate crude, acidified duck interferon: (a) Zn(COOCHah precipitation; (b) (NI-I4)2SO4 precipitation; (c) ultrafiltration through a P100 hollow fiber filter, cutoff 100,000 daltons, followed by concentration with a P5 hollow fiber filter, cutoff 5000 daltons; and (d) concentration with a P5 hollow fiber filter. The best method proved to be using a H1P5 hollow fiber filter with a 5000-dalton cutoff in an Amicon DC2 hollow fiber concentration system. This method permitted a 50- to 60-fold concentration in 36 hr at 4 ° with about 90% recovery of the original activity. The pH of the concentrate was raised to pH 7.2 by the dropwise addition of 1 M NaOH with stirring. One volume of buffer L containing 0.2/~g of phenylmethylsulfonylfluoride (PMSF) per milliliter was then added.
6 R. E. Smith,H. J. Zweerink,and W. K. Joklik, Virology39, 791 (1969).
568
PURIFICATION
AND CHARACTERIZATION
[79]
Column Chromatography of Concentrated Crude Duck Interferon. The following column materials, most of which have proved to be useful for purifying human or mouse interferon, were tested for their ability to aid in the purification of duck interferon: BioGel P-150, CM-Sephadex, phosphocellulose, Con A-Sepharose, SP-Sephadex, Sephacryl G-200, controlled-pore glass, and Sephadex G-75. None of these materials permitted the purification of duck interferon, usually because their use resulted either in poor separation of interferon from other proteins (resulting in a low purification factor) or in poor recovery of activity. The only column material that proved to be useful in the purification of duck interferon was a zinc chelate affinity column matrix. 7"8 This affinity chromatography method is based on the following principle: higher pH M - - L Me "+ + X P
lower pH
" M--L Me"+--XP
where M is the gel-forming matrix, L is a biscarboxymethyl amino group, Me "+ is a zinc ion (Zn~+), and XP is a protein molecule. The metal ions are thought to bind protein molecules through an interaction with the amino acids histidine and, probably, cysteine. This affinity matrix utilizes the ability of the metal ion to bind to these amino acid residues at a neutral or alkaline pH and to release them as the pH is lowered. The zinc chelate affinity gel matrix is prepared as follows. Epoxy-activated Sepharose 6B (15 ml) is washed for 1 hr with 1 liter of H20, dried by suction on a fritted-glass filter, and placed in a 50-ml round-bottom screwcap glass tube. A solution containing 2 g of iminodiacetic acid dissolved in 10 ml of 2 M Na~COa is added with thorough mixing; the tube is sealed with plastic wrap and incubated in a 65 ° bath with gentle shaking for 24 hr. The matrix is removed, placed onto a fritted-glass filter, washed briefly with H20, and dried by suction. It is then placed into another 50-ml tube containing 20 ml of 1 M ethanolamine; the tube is sealed as above and incubated in a 65 ° bath with gentle shaking for 4 hr. This treatment blocks any unreacted epoxy groups. The matrix is then removed, washed extensively with I-/20 on a fritted-glass filter, and stored in H20 at 4 °. A suspension of this matrix in H20 is then used to prepare 1.5 cm × 10 cm column beds (bed volume, 32 ml) in 1.5 cm × 20 cm Bio-Rad Econo-Columns. The matrix is converted to the zinc form by passing a 1% (w/v) ZnCI2 solution through the column. The column is washed extensively with 1-120 and equilibrated with buffer L. The column 7 j. Porath, J. Carlsson, I. Olsson, and G. Beifrago, Nature (London) 258, 598 (1975). s V. G. Edy, A. Billiau, and P. De Somer, J. Biol. Chem. 252, 5934 (1977).
[79]
DUCK FIBROBLAST INTERFERON
569
X
e
o
l',
z
i
o
l0
;'
•
ii Ii
6
z
2
~,-,
i - - --~-d-~e 20
40
60 80 1(30 FRACTION N U M B E R
120
140
160
FIG. 1. Zinc chelate affinity chromatography of concentrated crude duck interferon. Concentrated crude duck interferon (2.3 x 107 MTU in 65 ml) is neutralized to pH 7, diluted 1 : 1 with buffer L containing 0.2 p.g of phenylmethylsulfonyl fluoride PMSF, and is loaded onto a zinc chelate affinity chromatography column (1.5 x 18 cm column with a bed volume of about 32 ml) equilibrated with buffer L. Chromatography is carried out at 4 °. The column is washed with 300 ml of buffer WI, 200 ml of buffer W2, 300 ml of buffer W3, and 200 ml of buffer W4. Interferon is eluted with 80 ml of buffer El. The protein concentration of each fraction is determined by measuring the absorbance at 280 nm (OD~0m), and the interferon titer of each fraction is determined by the microtiter cytopathic effect reduction assay. Fractions 1 - 1 2 0 : 1 0 ml; fractions 121-170:2 ml. Protein, OD280,m, ($); interferon microtiter units (MTU/ml) (©).
can be regenerated with buffer E2 (which removes zinc ions), followed by extensive washing with H20. The matrix is then again converted to the zinc form as described above. After the column is loaded with crude interferon concentrate (see above), it is washed with 300 ml of buffer Wl, 200 ml of buffer W2, 300 ml of buffer W3,200 ml of buffer W4, and finally with 80 ml of buffer E1 which elutes the interferon (Fig. 1). The washes are collected in 10-ml fractions; the buffer E1 eluate, in 2-ml fractions. Polypropylene tubes are used throughout. About 90% of the interferon activity that is eluted from the column is contained in three fractions. Further chromatography of the peak interferon fractions on smaller zinc chelate affinity columns results in a further 2.5-fold purification; however, recovery of activity at this step is usually no more than 33%. The purification scheme for duck interferon is summarized in th~ table. The overall purification is about 3300-fold with approximately 809, recovery of initial activity.
570
[79]
PURIFICATION AND CHARACTERIZATION SUMMARY OF THE PURIFICATION OF DUCK INTERFERON Total
Total
Fraction
Volume a
protein b
interferon c
Specific activity a
Percent recovery e
Pooled crude interferon HIPS interferon concentrate Pooled peak zinc column fractions
3800
2660
2.5 × 107
9,400
100
65
ll00
2.3 × l07
21,000
92
4
0.64
2.0 x 107
3.1 x 10T
80
Purification factor f l 2.2 3300
a Volume in milliliters. b Total protein in milligrams as determined by Bio-Rad protein assay (duplicate assays). c Total interferon units as microtiter units (MTU). Specific activity in MTU of interferon per milligram of protein. Percentage recovery of the initial interferon activity (pooled crude interferon). Purification factor derived by dividing the final specific activity of a fraction by the initial specific activity of pooled crude interferon.
Estimate of the Purity of the Purified Duck Interferon Preparation Most estimates of specific activities of interferons that have been purified to homogeneity (human and mouse fibroblast and human leukocyte interferons) range from 2 to 5 x 10s units per milligram of protein. If duck fibroblast interferon possesses similar activity, the duck interferon preparation purified as described above would be about 10% pure. For comparison, the most highly purified chick fibroblast interferon preparation reported in the literature had a specific activity about ~ that of the duck interferon (1.6 x 106 units per milligram of protein). 9 When the duck interferon purified as described above is analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, two stainable bands are visible in positions corresponding to proteins migrating with apparent molecular weights of 17,500 and 20,000. Unfortunately the biological activity of the proteins in these bands cannot be determined directly, since duck interferon is inactivated by SDS.
a K. H. F a n t e s , J. Gen. Virol. 1, 257 (1967).
DUCK FIBROBLAST INTERFERON
2o3
different molecular species by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis is not feasible because type II interferon antiviral activity is irreversibly inactivated in the presence of SDS (unpublished observation). Antigenic Properties. As reported earlier, le'18 type II interferon cannot be neutralized by antisera directed against virus-induced interferon. The relatively large amounts of type II interferon prepared by the method described in this chapter made it possible to prepare a rabbit anti-type II mouse interferon serum. 11 This antiserum neutralized several T mitogeninduced interferons as well as the type II interferon that is induced in BCG-sensitized mice by injection of the specific antigen, tuberculin. Furthermore, the anti-type II interferon serum did not neutralize any of the type I interferons tested (virus-induced, lipopolysaccharide-induced, etc.). These results indicate that mitogen- and antigen-induced type II interferons are antigenically related, and confirm earlier observations about the different antigenic properties of type I and type II interferons. is j. Wietzerbin, R. Falcoff, L. Catinot, and E. Falcoff, Ann. lmmunol. (Paris) 128 C, 699 (1977).
[79] I n d u c t i o n
and Partial Purification of Duck Fibroblast Interferon
By ROBERT E. ZIEGLER and
WOLFGANG
K.
JOKLIK
Duck interferon is a useful reagent for studying the multiplication of RNA tumor viruses. Duck cells form a very useful system for this purpose for two reasons: they contain very little endogenous RNA tumor virus genetic information, certainly less than one complete genome; and certain cleavages of precursor to product proteins occur more slowly in duck embryo fibroblasts than in other cells. One example in this connection is the fact that RNA-dependent DNA polymerase can be isolated from RNA tumor viruses grown in duck embryo fibroblasts (DEF) not only in the a/3 and the a forms, but also in a/32 form, which is the precursor of the other two1; by contrast, very little if any of the/32 form can be isolated from RNA tumor viruses grown in chick embryo fibroblasts (CEF). Further, DEF form monolayers that are in many respects easier to handle and more uniform than CEF. It should be noted that chick interferon fails to protect DEF against A. Hizi and W. K. Joklik, J. Biol. Chem. 252, 2281 (1977).
METHODS IN ENZYMOLOGY, VOL. 78
Copyright © 1981by AcademicPress, Inc. All fights of reproduction in any form reserved. ISBN 0-12-18197g-7
564
PURIFICATION AND CHARACTERIZATION
[79]
viral infection; the titer of chick interferon on DEF is less than one-tenth the titer on CEF. Composition of Media, Buffers, and Solutions GM: Ham's F10, 10% (v/v) tryptose phosphate broth, 50 units of penicillin per milliliter, 50 ~g of streptomycin per milliliter, 2/zg of fungizone per milliliter, 5% calf serum (pH 7.2). PGM: GM plus 3% extra calf serum (total calf serum concentration equals 8%) and 2% chick serum (pH 7.2) LSMG: GM with only 0.1% (v/v) calf serum instead of 5% OLMC: 2x GM, 50/zg of gentamycin per milliliter PBSI: 0.14 M NaC1, 8 mM Na~HPO4, 1.5 mM KH2PO4, 2.5 mM KC1 (pH 7.0) Puck's saline A: 0.8% NaC1, 0.04% KCI, 0.009% Na~HPO4.H~O, 0.006% KHzPO4, 0.1% glucose, 0.035% NaHCOs, 0.4% MgClz.6 H~O, 0.002% phenol red (pH 7.2) Buffer L: 150 mM NaC1, 50 mM sodium phosphate (pH 7.2) Buffer Wl: Same as buffer L Buffer W2:0.40 M NaC1, 50 mM sodium phosphate (pH 7.2) Buffer W3:0.60 M NaC1, 50 mM sodium phosphate (pH 6.5) Buffer W4:0.80 M NaC1, 50 mM NaCOOCH3 (pH 5.5) Buffer El: 0.80 M NaCI, 50 mM NaCOOCHs (pH 4.5) Buffer E2:0.50 M NaCI, 50 mM EDTA Preparation of Duck E m b r y o Fibroblasts Primary DEF are prepared from 12- to 13-day-old Peking duck embryos by a modification of the method of Vogt. 2 Briefly, after removing, under sterile conditions, the shell and outer membrane, embryos are removed from the eggs with a sterile rectal hook and are placed in a 150-mm plastic tissue culture dish containing sterile PBS1. The embryos are decapitated and eviscerated using sterile forceps, rinsed in a second tissue culture dish containing PBS1, and placed into a third dish containing fresh PBS1. This is repeated for 15-20 embryos per preparation. A 0.25% (v/v) trypsin solution, 10 ml per embryo, is added to a 300-ml trypsinizing flask containing a magnetic stirring bar. The embryos are placed into a sterile 10-ml glass syringe fitted with a 1.5-inch, 16-gauge needle and are then passed through the needle into the trypsinizing flask. Ten embryos are trypsinized in each 300-ml flask. 2 p. K. Vogt, in "Fundamental Techniques in Virology " (K. Habel and N. P. Salzman, eds.), p. 198. Academic Press, New York, 1969.
[79]
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565
The trypsin-embryo suspension is stirred for 15 min at 37°, the flask is tilted, the larger debris are allowed to settle, and the supernatant containing the trypsinized cells is poured sterilely into centrifuge bottles containing 20 ml of cold PGM per embryo. If the extent of digestion of the embryos is insufficient, the trypsinization steps are repeated. The cells are pelleted (1500 rpm, 15 min, 4°), the supernatant is discarded, and the cell pellet is resuspended in warm PGM (10 ml per embryo). The cells are counted, after diluting 0.2 ml of the cell suspension into 10 ml of PBS1, with a Coulter counter Model FN with an attenuation setting of 1, an aperture setting of 128, and a threshold setting of 25. The cells are seeded into 150-mm plastic tissue culture dishes in 25 ml of PGM (1.2 × 107 cells/dish) or into 1330-cm~ glass roller culture bottles in 200 ml of PGM (2.5 × 108 cells/roller bottle). Tissue culture dishes are incubated at 37° in a water-jacketed incubator in a 5% CO2/95% air atmosphere. Roller culture bottles are placed onto a Bellco roller bottle apparatus in a 37° warm room and are rotated at 6 revolutions/hr. Monolayers are confluent after 48 hr. Assay of Duck Interferon Duck interferon is assayed either by a microtiter cytopathic effect (CPE) reduction or by a plaque reduction assay, in both cases with vesicular stomatitis virus (VSV) grown in DEF. It should be noted that VSV grown in CEF is essentially incapable of infecting DEF. Microtiter CPE Reduction Assay. Duck interferon is assayed by a CPE reduction assay combining the techniques of Henderson and Joklik 3 and Tilles and Finland. 4 Briefly, 100/zl of GM are added to each well of a Linbro microtiter 96-weU tissue culture plate (8 rows of 12 wells each) with an 8 × 100 Izl Titertek manifold. One hundred microliters of each duck interferon sample to be assayed is added to the first well of each row, and simultaneous serial twofold dilutions are performed with an 8 x 10/xl manifold, a 100-/zl row being discarded at the end of the last dilution (final volume, 100/zl/well). To each well 4 × 104 DEF are added in 50/zl of GM with an 8 × 50/zl manifold, and the plates are incubated for 24 hr at 37°, during which time the cells attach to the surface of the well to form monolayers and, if interferon is present, become resistant to virus challenge. The monolayers are challenged with 200 plaque-forming units (PFU) of VSV per well in 50/zl of GM at the end of the 24-hr incubation period. The plates are incubated for an additional 36 hr at 37°, and the monolayers are then examined under a light microscope for evidence of 3 D. R. H e n d e r s o n and W. K. Joklik, Virology 91, 389 (1978). 4 j. G. Tiiles and M. Finland, Appl. Microbiol. 16, 1706 (1968).
566
PURIFICATION AND CHARACTERIZATION
[79]
virus-induced CPE. Control monolayers with no duck interferon added show complete destruction of cell monolayers. In interferon-treated cell monolayers, the change from complete protection to complete destruction of the cell monolayer usually occurs within a range of three consecutive wells. The titer of an interferon sample in microtiter units (MTU) is defined as the reciprocal of that dilution which is halfway between the highest dilution of interferon that completely protects the cell monolayer and the lowest dilution of interferon that allows the complete destruction of the cell monolayer. Plaque Reduction Assay. The assay used is that of Lai and Joklik. 5 D E F are seeded into 60-mm tissue culture dishes at a density of 2 x 106 cells/dish. When the monolayers are confluent, the medium is removed from the dishes, and 3-ml aliquots of serial dilutions in GM of interferon preparations are added to duplicate dishes. The dishes are incubated for 24 hr at 37°, the interferon-containing medium or control medium is removed, 100 PFU of VSV are added to each dish in 0.1 ml of GM, the dishes are incubated at 37° for I hr, and each dish is then overlaid with 5 ml of a mixture of 41% (v/v) prewarmed agar and 59% (v/v) OLMC. The overlay is allowed to harden at room temperature, and the dishes are incubated at 37° until plaques are visible on control plates by light microscopy (about 36 hr.) One milliliter of a neutral red solution is then added to each dish, the dishes are incubated for an additional 6 hr at 37°, and the plaques are counted using a light box with indirect illumination. One plaque-reduction unit of duck interferon (1 PRDs0) is defined as that dilution of a standard duck interferon preparation that reduces the number of plaques developing in an interferon-treated DEF monolayer to 50% of the number of plaques developing in the control monolayer. Under these conditions 1 MTU is equivalent to 2 PRDs0. Induction of Duck Interferon The optimum inducer of interferon in DEF is reovirus. DEF are nonpermissive for reovirus. The effect of several variables on the yield of duck interferon were studied; among them were (a) the effect of varying reovirus multiplicity; (b) the effect of irradiating reovirus with various doses of ultraviolet irradiation; (c) the effect of varying the length of the incubation period following the initial induction period; (d) the effect of varying the concentration of serum in the incubation medium; and (e) the effect of varying the incubation medium volume. As a result of these studies, the most efficient method of inducing the synthesis of interferon in confluent monolayers of DEF has been determined to be as follows. s M.-T. Lai and W. K. Joklik,
Virology 51, 191 (1973).
[79]
DUCK FIBROBLAST INTERFERON
567
Medium is removed from confluent monolayers of DEF (2.5 x l08 cells) growing in 1330-cm 2 glass roller culture bottles, and the cells are inoculated with 50,000 particles (about 250 PFU)/ceU of reovirus serotype 3 (strain Dearing) 6 suspended in 10 ml of a 1 : 1 mixture of Puck's saline A and LSGM. After 2 hr at 37°, 40 ml of LSGM are added, and the bottles are incubated for 58 hr at 37°, when the interferon-containing medium is harvested. This induction process yields about 2.5 x 104 MTU of interferon per 107 cells (about 6 x 105 MTU per bottle). Purification of Duck Interferon
General Considerations. The purification of duck interferon is divided into three steps: (a) an acidification and clarification step; (b) a concentration step; and (c) a column chromatography step. Each step is discussed below. Duck interferon has several properties that affect the manner in which it can be purified: (a) it is sensitive to dialysis, more so with increasing purity; (b) it is rapidly inactivated by exposure to sodium dodecyl sulfate (SDS) in concentrations of 0.01-1.0% (v/v) and to 0.01% (v/v) 2-mercaptoethanol (2-ME); and (c) it adsorbs readily to glass and must therefore be stored in polypropylene containers. Initial Processing of Crude Duck Interferon. The harvested medium containing crude interferon is centrifuged to remove cellular debris (2000 rpm, 20 min, 4°) and acidified to pH 2; it can then be stored at 4° for at least 2 months without loss of activity. The acidified, crude interferon preparation is then centrifuged at 40,000 rpm for 45 rain at 4° to remove residual reovirus, precipitated protein, and other debris. Concentration of Crude Duck Interferon. Several methods were tested for their ability to concentrate crude, acidified duck interferon: (a) Zn(COOCHah precipitation; (b) (NI-I4)2SO4 precipitation; (c) ultrafiltration through a P100 hollow fiber filter, cutoff 100,000 daltons, followed by concentration with a P5 hollow fiber filter, cutoff 5000 daltons; and (d) concentration with a P5 hollow fiber filter. The best method proved to be using a H1P5 hollow fiber filter with a 5000-dalton cutoff in an Amicon DC2 hollow fiber concentration system. This method permitted a 50- to 60-fold concentration in 36 hr at 4 ° with about 90% recovery of the original activity. The pH of the concentrate was raised to pH 7.2 by the dropwise addition of 1 M NaOH with stirring. One volume of buffer L containing 0.2/~g of phenylmethylsulfonylfluoride (PMSF) per milliliter was then added.
6 R. E. Smith,H. J. Zweerink,and W. K. Joklik, Virology39, 791 (1969).
568
PURIFICATION
AND CHARACTERIZATION
[79]
Column Chromatography of Concentrated Crude Duck Interferon. The following column materials, most of which have proved to be useful for purifying human or mouse interferon, were tested for their ability to aid in the purification of duck interferon: BioGel P-150, CM-Sephadex, phosphocellulose, Con A-Sepharose, SP-Sephadex, Sephacryl G-200, controlled-pore glass, and Sephadex G-75. None of these materials permitted the purification of duck interferon, usually because their use resulted either in poor separation of interferon from other proteins (resulting in a low purification factor) or in poor recovery of activity. The only column material that proved to be useful in the purification of duck interferon was a zinc chelate affinity column matrix. 7"8 This affinity chromatography method is based on the following principle: higher pH M - - L Me "+ + X P
lower pH
" M--L Me"+--XP
where M is the gel-forming matrix, L is a biscarboxymethyl amino group, Me "+ is a zinc ion (Zn~+), and XP is a protein molecule. The metal ions are thought to bind protein molecules through an interaction with the amino acids histidine and, probably, cysteine. This affinity matrix utilizes the ability of the metal ion to bind to these amino acid residues at a neutral or alkaline pH and to release them as the pH is lowered. The zinc chelate affinity gel matrix is prepared as follows. Epoxy-activated Sepharose 6B (15 ml) is washed for 1 hr with 1 liter of H20, dried by suction on a fritted-glass filter, and placed in a 50-ml round-bottom screwcap glass tube. A solution containing 2 g of iminodiacetic acid dissolved in 10 ml of 2 M Na~COa is added with thorough mixing; the tube is sealed with plastic wrap and incubated in a 65 ° bath with gentle shaking for 24 hr. The matrix is removed, placed onto a fritted-glass filter, washed briefly with H20, and dried by suction. It is then placed into another 50-ml tube containing 20 ml of 1 M ethanolamine; the tube is sealed as above and incubated in a 65 ° bath with gentle shaking for 4 hr. This treatment blocks any unreacted epoxy groups. The matrix is then removed, washed extensively with I-/20 on a fritted-glass filter, and stored in H20 at 4 °. A suspension of this matrix in H20 is then used to prepare 1.5 cm × 10 cm column beds (bed volume, 32 ml) in 1.5 cm × 20 cm Bio-Rad Econo-Columns. The matrix is converted to the zinc form by passing a 1% (w/v) ZnCI2 solution through the column. The column is washed extensively with 1-120 and equilibrated with buffer L. The column 7 j. Porath, J. Carlsson, I. Olsson, and G. Beifrago, Nature (London) 258, 598 (1975). s V. G. Edy, A. Billiau, and P. De Somer, J. Biol. Chem. 252, 5934 (1977).
[79]
DUCK FIBROBLAST INTERFERON
569
X
e
o
l',
z
i
o
l0
;'
•
ii Ii
6
z
2
~,-,
i - - --~-d-~e 20
40
60 80 1(30 FRACTION N U M B E R
120
140
160
FIG. 1. Zinc chelate affinity chromatography of concentrated crude duck interferon. Concentrated crude duck interferon (2.3 x 107 MTU in 65 ml) is neutralized to pH 7, diluted 1 : 1 with buffer L containing 0.2 p.g of phenylmethylsulfonyl fluoride PMSF, and is loaded onto a zinc chelate affinity chromatography column (1.5 x 18 cm column with a bed volume of about 32 ml) equilibrated with buffer L. Chromatography is carried out at 4 °. The column is washed with 300 ml of buffer WI, 200 ml of buffer W2, 300 ml of buffer W3, and 200 ml of buffer W4. Interferon is eluted with 80 ml of buffer El. The protein concentration of each fraction is determined by measuring the absorbance at 280 nm (OD~0m), and the interferon titer of each fraction is determined by the microtiter cytopathic effect reduction assay. Fractions 1 - 1 2 0 : 1 0 ml; fractions 121-170:2 ml. Protein, OD280,m, ($); interferon microtiter units (MTU/ml) (©).
can be regenerated with buffer E2 (which removes zinc ions), followed by extensive washing with H20. The matrix is then again converted to the zinc form as described above. After the column is loaded with crude interferon concentrate (see above), it is washed with 300 ml of buffer Wl, 200 ml of buffer W2, 300 ml of buffer W3,200 ml of buffer W4, and finally with 80 ml of buffer E1 which elutes the interferon (Fig. 1). The washes are collected in 10-ml fractions; the buffer E1 eluate, in 2-ml fractions. Polypropylene tubes are used throughout. About 90% of the interferon activity that is eluted from the column is contained in three fractions. Further chromatography of the peak interferon fractions on smaller zinc chelate affinity columns results in a further 2.5-fold purification; however, recovery of activity at this step is usually no more than 33%. The purification scheme for duck interferon is summarized in th~ table. The overall purification is about 3300-fold with approximately 809, recovery of initial activity.
570
[79]
PURIFICATION AND CHARACTERIZATION SUMMARY OF THE PURIFICATION OF DUCK INTERFERON Total
Total
Fraction
Volume a
protein b
interferon c
Specific activity a
Percent recovery e
Pooled crude interferon HIPS interferon concentrate Pooled peak zinc column fractions
3800
2660
2.5 × 107
9,400
100
65
ll00
2.3 × l07
21,000
92
4
0.64
2.0 x 107
3.1 x 10T
80
Purification factor f l 2.2 3300
a Volume in milliliters. b Total protein in milligrams as determined by Bio-Rad protein assay (duplicate assays). c Total interferon units as microtiter units (MTU). Specific activity in MTU of interferon per milligram of protein. Percentage recovery of the initial interferon activity (pooled crude interferon). Purification factor derived by dividing the final specific activity of a fraction by the initial specific activity of pooled crude interferon.
Estimate of the Purity of the Purified Duck Interferon Preparation Most estimates of specific activities of interferons that have been purified to homogeneity (human and mouse fibroblast and human leukocyte interferons) range from 2 to 5 x 10s units per milligram of protein. If duck fibroblast interferon possesses similar activity, the duck interferon preparation purified as described above would be about 10% pure. For comparison, the most highly purified chick fibroblast interferon preparation reported in the literature had a specific activity about ~ that of the duck interferon (1.6 x 106 units per milligram of protein). 9 When the duck interferon purified as described above is analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, two stainable bands are visible in positions corresponding to proteins migrating with apparent molecular weights of 17,500 and 20,000. Unfortunately the biological activity of the proteins in these bands cannot be determined directly, since duck interferon is inactivated by SDS.
a K. H. F a n t e s , J. Gen. Virol. 1, 257 (1967).