- Email: [email protected]
[69] Purification of human leukocyte interferon by two-dimensional polyacrylamide gel electrophoresis
[69]
T W O - D I M E N S I O N A L GEL PURIFICATION
481
from I to 4 × l0 s reference units/mg on the MDBK cell line, and 0.5 to 4 × los reference units/mg on human AG-1732 cells (not shown). The overall yield of interferon is about 20-25%, with yields of each species ranging from 0.2 to 11%. Polyacrylamide gel electrophoresis (Fig. 5) demonstrates the purity of these interferons. All protein bands on the gel have antiviral activity, except the minor 12,000-dalton contaminants of ca and b~, and the minor 23,000-dalton contaminant ofb~. Molecular weights and amino acid compositions of the interferons are summarized in Tables II and III. Although amino acid compositions of the interferons are very similar, the species can be distinguished by differences in molecular weight, activity on MDBK and AG-1732 cells, and separation by HPLC. Correlation of KG-1 interferons with those purified from leukocytes from patients with chronic myelogenous leukemia 4 is currently under way. Acknowledgments The authors wish to thank Philip FamiUetti, Lauren Costello, Cynthia Rose, Eileen Gusciora, and Diane Copsey for production and maintenance of the KG-1 cells; Joseph Bigley and Cynthia Rose for performing the antiviral assays of the intefferons. We also wish to express our gratitude to Larry Brink for carrying out the amino acid analyses.
[69] Purification of Human Leukocyte Interferon by Two-Dimensional Polyacrylamide (3el E l e c t r o p h o r e s i s By LEO S. LIN and WILLIAM E. STEWART II
Interferons were described in the late 1950s by Isaacs et al. 1,2 as substances released by virus-infected cells that could confer antiviral resistance to cells. Since then, a number of other biological activities have been ascribed to interferon (see other sections of Vols. 78 and 79 for details). In the absence of homogeneous interferons, the ascribing of these other biological activities to interferon have often been a source of controversy. In recent years, the availability of large quantities of interferons via varied forms of large-scale production methods (see Section II of this volume), and the observations that various interferon activities can be recovered after sodium dodecyl sulfate-polyacrylamide gel electrophoresis ( S D S i A. Isaacs and J. Lindenmann, Prec. R. Soc. London, Ser. B 147, 258 (1957), A, Isaacs, J. Lindenmann, and R. C. Valentine, Prec. R. Soc. London, Set. B 147, 268 (1957).
METHODS IN ENZYMOLOGY, VOL. 78
Copyright © 1981by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181978-7
482
PURIFICATION AND CHARACTERIZATION
[69]
PAGE) a'4 have greatly accelerated the progress of purification and characterization of interferons. Human leukocyte interferon can be described as a heterogeneous mixture of proteins containing at least two major species of molecules having peaks of activities at 21,000 daltons and 18,000-15,000 daltons when analyzed by SDS-PAGE, 4 and extensive heterogeneity with isoelectric points ranging from pH 5.5 to pH 7.0 when analyzed by analytical isoelectric focusing. 5 These marked heterogeneities of human leukocyte interferon forms, contributed at least in part by carbohydrate moieties, have hindered isolation of homogeneous pure forms of this material. In order to obtain homogeneous human leukocyte interferon we have devised a purification scheme that includes the elimination of heterogeneity by chemical oxidation with sodium periodate, 5 molecular sieve column chromatography, affinity column chromatography, and a modification of the two-dimensional gel electrophoresis technique of O'Farrell. e Homogeneous spots of two forms of human leukocyte interferons are obtained with this method, each with a final specific activity of about 109 units per milligram of protein. Materials and Apparatus Human leukocyte interferon preparations purified to a specific activity of about 10e units per milligram of protein (PIF-A) were obtained from Dr. Kari Cantell (Central Public Health Laboratories, Helsinki, Finland). Chromatographic resins Sephadex G-100 and poly(U)-Sepharose were obtained from Pharmacia Fine Chemicals (Piscataway, N J). Carrier ampholytes for isoelectric focusing were purchased from LKB Instruments (RockviUe, MD). Isoelectric focusing was performed in a Buchler Polyanalyst electrophoresis chamber (Fort Lee, N J), and focusing gels were removed with a gel extruder (LAC Enterprise, P.O. Box 301, Santa Barbara, CA). SDS-PAGE was performed in a slab gel electrophoresis apparatus designed to accommodate gel plates 35 cm wide and 30 cm tall. Acrylamide, bisacrylamide, TEMED, SDS, all electrophoretic grade, were obtained from Bio-Rad (Richmond, CA). Protein concentrations are determined either by the dye binding method with the Bio-Rad protein assay kit 7 or fluorometric assay with Fluram (Roche Diagnostics, Nutley, 3 W. E. Stewart II, Virology 61, 81 (1974). 4 W. E. Stewart II and J. Desmyter, Virology 67, 68 (1975). 5 W. E. Stewart II, L. S. Lin, M. Wiranowska-Stewart, and K. Cantell, Proc. Natl. Acad. Sci. U.S.A. 74, 4200 (1977). 8 p. H. O'Farrell, J. Biol. Chem. 250, 4007 (1975). M. M. Bradford, Anal. Biochem. 72, 248 (1976).
[69]
TWO-DIMENSIONAL GEL PURIFICATION
483
N J). s Nonidet P-40 (NP-40) was from Shell Imperial (UK) and ultrapure urea was from Schwarz-Mann (Orangeburg, NY). All other chemicals were reagent grade.
Reagents NalO4 (meta), 0.02 M, in 0.1 M sodium acetate, pH 4.5 Ethylene glycol, 50% (v/v) NI-I4HCO3,0.03 M, pH 7.6 Phosphate-buffered saline, pH 7.5 Tris • HC1, 0.01 M, pH 7.5 Tris • HCI, 0.01 M and 1.0 M NaC1, pH 7.5 Na2HPO4/NaPO4,0.1 M, pH 7.2 NaCI, 0.0375 M Experimental Procedures Partially purified human leukocyte interferon preparation, containing major protein contaminants at about 67,000 daltons and 25,000 daltons (Fig. 1), is diluted 1:2 (v/v) with phosphate-buffered saline and then mixed with an equal volume of periodate buffer (0.02 M NaIO4 in 0. l M sodium acetate, pH 4.5). The mixture is vortexed, and the reaction mixture is stored in the dark at 4 ° for 2-4 hr. After the desired period of treatment the mixture is centrifuged at 13,000 g for l0 rain. The precipitate formed during the reaction contains most of the 25,000 dalton protein bul little of the interferon activity. The supernatant fluid, which contains the 67,000 dalton contaminant and most of the interferon activity, is decanted and diluted 1 : 10 (v/v) with 50% ethylene glycol in water to stop the reaction. The preparation is then dialyzed extensively against 0.03 34 NI-I4HCO3 buffer (pH 7.6), and lyophilized. The lyophilized sample is resuspended in a minimum volume of Tris buffer (0.01 M, pH 7.5), and applied to a Sephadex G-100 column (2.6 cm in diameter by 170 cm in length) equilibrated with the same buffer. Samples are eluted with Tris buffer at a flow rate of 1 ml/min, and 10-ml fractions are collected with an LKB Ultrorac Model 7000 fraction collector. Protein concentrations are monitored with a Pharmacia UV I monitor operating at 280 nm with 0.05 to 0.1 absorbance unit full scale (AUFS). Interferon activities are determined by the cytopathic effect inhibition assay2 Fractions containing peak interferon activities well separated from the 67,000 dalton protein contaminant are pooled and applied to a poly(U)8 S. Udenfriend, S. Stein, P. BOhlen, W. Dairman, W. Leimgruber, and M. Weigele, Scie n c e 178, 871 (1972). 9 W. E. Stewart II, "The Interferon System." Springer-Verlag, Vienna and New York, 1979.
484
[69]
PURIFICATION AND CHARACTERIZATION
mol. wt. column marke,,r,S e!uent
.
104 ppt.
"
PIF.
,
l x 1 0 s Interferon units per sample FIG. I. One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of human leukocyte interferon demonstrating the selective removal of the 25,000-dalton noninterferon protein by periodate precipitation and the separation of the 67,000-dalton contaminant protein by gel filtration. Left-hand track represents the protein
[69]
TWO-DIMENSIONAL GEL PURIFICATION
485
Sepharose column (with a bed dimension of 1.6 cm in diameter by 1.5 cm height) equilibrated with the same Tris buffer. Protein concentration is monitored with a Pharmacia UV I monitor operating at 280 nm with 0.01 AUFS. After the samples are applied onto the poly(U)-Sepharose, the column is washed with the same Tris buffer until no detectable protein is further eluted from the column. The column is then eluted with 0.01 M Tris-l.0 M NaCI, pH 7.5. Four-milliliter fractions are collected with a Gilson microfractionator. Fractions are assayed for interferon activities and then pooled, dialyzed against 0.03 M NH4HCO3 buffer, and then lyophilized for application to isoelectric focusing gels.
Two-Dimensional Gel Electrophoresis A modified O'Farrcll two-dimensional gel clectrophoresis system is used to further purify the human leukocyte interferon preparations. The modifications are (a) the omission of reducing agents, such as/3-mcrcaptoethanol, in all solutions; and (b), the increased length of thc isoelectric focusing gel from l I c m to 24 c m with the corresponding increase of the second-dimension S D S gel slab to 35 c m wide by 30 c m in height. Isoclectric focusing is carried out in long capillary gels (2.8 m m in diameter by 24 c m in length). The gels contain 9 M urea, 4 % acrylamide, 0.2% bisacrylamide, 2 % NP-40 (w/v) and 2 % Ampholine (v/v) (LKB, 4: l mixture ofpH 5-7 and p H 3-10). Polymerization is initiatedby addition of T E M E D (7 tzl/10 ml) and a m m o n i u m persulfatc (I txg/10 ml). The gel solution is introduced into the gel tubes with capillary tubing connected via a hypodermic needle to a syringe. The filledgel tubes arc further degassed to assure uniformity. The gels were then processed and prefocused as described by O'Farcll.6 Interferon samples are solubilized in a minimum volume of focusing sample buffer, which contains 9.5 M urea, 2 % (w/v) NP-40 and 2 % A m pholine mixture to a finalprotein concentration of approximately 1/zg//zl; 25-35/zl of solubilized interfcron sample is applied to each isoelcctricfocusing gel. Focusing is carried out at 400 V constant voltage until a minim u m current is obtained. The focusing voltage is then raised to 800 V for markers; each of the other tracks in the slab gel was loaded with approximately the same total units of interferon activity: PIF, partially purified human leukocyte interferon with a specific activity of about 108units per milligramof protein (starting material); IO~ ppt, precipitate formed after periodate treatment, composed of essentially the 25,000-dalton contaminant and very little interferon activity; IO~ sup, supernatant material after periodate treatment, containing most of the interferon activity and the 67,000-dalton contaminant, but devoid of the 25,000dalton species; Sephadex G-100 column eluent: fractions containingpeak interferon activity arefree of the bulk of both the 67,000-dalton and 25,000-dalton contaminant proteins.
486
[69]
PURIFICATION AND CHARACTERIZATION Isoetectric M48,"10
w
a
Focusing pH4.03
•
•
eTK
e
~) 45K
Q
o 30K
m
h 20cm
~
m 14AK 24cm
FIG. 2. Two-dimensional gel electrophoresis of human leukocyte interferon. A two-dimensional polyacrylamide slab of the periodate-precipitated, Sephadex G-100-separated, and poly(U)-Sepharose-chromatographed human leukocyte interferon was stained for protein with Coomassie Brilliant Blue. The two spots of interferon antiviral activities determined by assaying fractions from a duplicate unstained gel correspond to the two stained spots (enclosed area is enlarged and inserted at upper right). The spot on the acidic side corresponds to the interferon species, which has nearly equal antiviral activities on both human and bovine cells, whereas the spot on the basic side corresponds to the interferon species with predominant antiviral activities on bovine ceils.
60 rain before termination. Gels are extruded onto sheets of parafilm with a gel extruder (LAC Enterprise). Gels are immediately frozen at - 70 ° and fractionated into 2.2-ram segments, and each segment is placed in 1 ml of 1% SDS and 0.15 M NaC1 solution and stored for elution and assay. The pH gradient is determined by eluting 1-cm fractions of a gel in 1 ml of 0.0375 M NaC1 overnight and then measuring the pH of the solution at room temperature. Electrophoresis in the second dimension is carded out on slab gels (35 cm × 30 cm x 0.15 cm) with single 30-cm slot. The gel contains 0.45 M urea, 10% acrylamide, 0.8% bisacrylamide, 0.1 M phosphate buffer, pH 7.2, 0.1% SDS. Polymerization is initiated by addition of TEMED (6/zl/10 ml) and ammonium persulfate (0.8 mg/ml). Isoelectric focusing gels are embedded onto the SDS gel slab by molten agarose solution (1% agarose, 2% SDS in 0.1 M sodium phosphate, pH 7.2). The upper buffer is 2% SDS in 0.1 M sodium phosphate, pH 7.2, and the lower buffer is 0.1% SDS in 0.1 M sodium phosphate, pH 7.2. Electrophoresis is at 15 W constant power toward the anode. After electrophoresis for 20 rain,
[70]
ANTIBODY AFFINITY CHROMATOGRAPHY
487
the top electrode buffer is removed and replaced with I ml of 0.1% SDS in 0.1 M sodium phosphate buffer, pH 7.5. Electrophoresis is terminated when the tracking dye bromophenol blue has migrated about 23 cm through the gel. The slab gel is removed and either stained for protein with Coomassie Brilliant Blue or the appropriate area is cut into fractions of 0.5-cm squares and eluted with 1 ml of 1% SDS in 0.15 M NaCI and assayed for interferon activity on both human and bovine cell cultures. 1° This method of two-dimensional gel electrophoresis of periodatetreated preparations can be used to isolate two distinct species of human leukocyte interferon molecules differing in human to bovine activity ratios (Fig. 2). The extra gel lengths employed in both the isoelectric focusing dimension and SDS-PAGE dimension is necessary to resolve completely the various species of human leukocyte interferon. l0 L. S. Lin, M. Wiranowska-Stewart, T. Chudzio, and W. E. Stewart II, J. Gen. Virol. 39, 125 (1978).
[70] A n t i b o d y By
Affinity Chromatography Leukocyte Interferon
of Human
K U R T BERG a n d IVER HERON
In theory, the antibody affinity chromatography technique is very simple for obtaining pure human leukocyte interferon proteins, provided that only strictly monospecific anti-interferon immunoglobulins are used and no fortuitous (nonspecific) immunological crossing-over occurs. Accordingly, the immunoglobulins are immobilized by covalently binding to a suitable matrix, for example, Sepharose 4B, without destroying or distorting the antigenic sites, and a small column is prepared. At neutral pH (pH 6.5-7.5) the immobilized antibodies will bind only the interferon proteins when a crude preparation of human leukocyte interferon is passed through the column. After a thorough wash the interferon is released by disrupting the antigen-antibody complex in a suitable manner, such as by lowering the pH (to 2.4), and pure interferon proteins are obtained with a high recovery (75-100%). The term "monospecific antibodies" implies, in this context, that the anti-interferon immunoglobulin preparation reacts only with the interferon proteins, n o t with any of the contaminating proteins present in an overwhelming amount in crude interferon preparations. 1'2 To compeni K. Berg, Scand. J. lmmunol. 6, 77 (1977). z K. Berg, I. Heron, and R. Hamilton, Stand. J. lmmunol. 8, 429 (1978).
METHODS IN ENZYMOLOGY, VOL. 78
Copyright © 1981 by Academic Press, Inc. All fights of reproduction in any form reserved. ISBN 0-12-181978-7
T W O - D I M E N S I O N A L GEL PURIFICATION
481
from I to 4 × l0 s reference units/mg on the MDBK cell line, and 0.5 to 4 × los reference units/mg on human AG-1732 cells (not shown). The overall yield of interferon is about 20-25%, with yields of each species ranging from 0.2 to 11%. Polyacrylamide gel electrophoresis (Fig. 5) demonstrates the purity of these interferons. All protein bands on the gel have antiviral activity, except the minor 12,000-dalton contaminants of ca and b~, and the minor 23,000-dalton contaminant ofb~. Molecular weights and amino acid compositions of the interferons are summarized in Tables II and III. Although amino acid compositions of the interferons are very similar, the species can be distinguished by differences in molecular weight, activity on MDBK and AG-1732 cells, and separation by HPLC. Correlation of KG-1 interferons with those purified from leukocytes from patients with chronic myelogenous leukemia 4 is currently under way. Acknowledgments The authors wish to thank Philip FamiUetti, Lauren Costello, Cynthia Rose, Eileen Gusciora, and Diane Copsey for production and maintenance of the KG-1 cells; Joseph Bigley and Cynthia Rose for performing the antiviral assays of the intefferons. We also wish to express our gratitude to Larry Brink for carrying out the amino acid analyses.
[69] Purification of Human Leukocyte Interferon by Two-Dimensional Polyacrylamide (3el E l e c t r o p h o r e s i s By LEO S. LIN and WILLIAM E. STEWART II
Interferons were described in the late 1950s by Isaacs et al. 1,2 as substances released by virus-infected cells that could confer antiviral resistance to cells. Since then, a number of other biological activities have been ascribed to interferon (see other sections of Vols. 78 and 79 for details). In the absence of homogeneous interferons, the ascribing of these other biological activities to interferon have often been a source of controversy. In recent years, the availability of large quantities of interferons via varied forms of large-scale production methods (see Section II of this volume), and the observations that various interferon activities can be recovered after sodium dodecyl sulfate-polyacrylamide gel electrophoresis ( S D S i A. Isaacs and J. Lindenmann, Prec. R. Soc. London, Ser. B 147, 258 (1957), A, Isaacs, J. Lindenmann, and R. C. Valentine, Prec. R. Soc. London, Set. B 147, 268 (1957).
METHODS IN ENZYMOLOGY, VOL. 78
Copyright © 1981by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181978-7
482
PURIFICATION AND CHARACTERIZATION
[69]
PAGE) a'4 have greatly accelerated the progress of purification and characterization of interferons. Human leukocyte interferon can be described as a heterogeneous mixture of proteins containing at least two major species of molecules having peaks of activities at 21,000 daltons and 18,000-15,000 daltons when analyzed by SDS-PAGE, 4 and extensive heterogeneity with isoelectric points ranging from pH 5.5 to pH 7.0 when analyzed by analytical isoelectric focusing. 5 These marked heterogeneities of human leukocyte interferon forms, contributed at least in part by carbohydrate moieties, have hindered isolation of homogeneous pure forms of this material. In order to obtain homogeneous human leukocyte interferon we have devised a purification scheme that includes the elimination of heterogeneity by chemical oxidation with sodium periodate, 5 molecular sieve column chromatography, affinity column chromatography, and a modification of the two-dimensional gel electrophoresis technique of O'Farrell. e Homogeneous spots of two forms of human leukocyte interferons are obtained with this method, each with a final specific activity of about 109 units per milligram of protein. Materials and Apparatus Human leukocyte interferon preparations purified to a specific activity of about 10e units per milligram of protein (PIF-A) were obtained from Dr. Kari Cantell (Central Public Health Laboratories, Helsinki, Finland). Chromatographic resins Sephadex G-100 and poly(U)-Sepharose were obtained from Pharmacia Fine Chemicals (Piscataway, N J). Carrier ampholytes for isoelectric focusing were purchased from LKB Instruments (RockviUe, MD). Isoelectric focusing was performed in a Buchler Polyanalyst electrophoresis chamber (Fort Lee, N J), and focusing gels were removed with a gel extruder (LAC Enterprise, P.O. Box 301, Santa Barbara, CA). SDS-PAGE was performed in a slab gel electrophoresis apparatus designed to accommodate gel plates 35 cm wide and 30 cm tall. Acrylamide, bisacrylamide, TEMED, SDS, all electrophoretic grade, were obtained from Bio-Rad (Richmond, CA). Protein concentrations are determined either by the dye binding method with the Bio-Rad protein assay kit 7 or fluorometric assay with Fluram (Roche Diagnostics, Nutley, 3 W. E. Stewart II, Virology 61, 81 (1974). 4 W. E. Stewart II and J. Desmyter, Virology 67, 68 (1975). 5 W. E. Stewart II, L. S. Lin, M. Wiranowska-Stewart, and K. Cantell, Proc. Natl. Acad. Sci. U.S.A. 74, 4200 (1977). 8 p. H. O'Farrell, J. Biol. Chem. 250, 4007 (1975). M. M. Bradford, Anal. Biochem. 72, 248 (1976).
[69]
TWO-DIMENSIONAL GEL PURIFICATION
483
N J). s Nonidet P-40 (NP-40) was from Shell Imperial (UK) and ultrapure urea was from Schwarz-Mann (Orangeburg, NY). All other chemicals were reagent grade.
Reagents NalO4 (meta), 0.02 M, in 0.1 M sodium acetate, pH 4.5 Ethylene glycol, 50% (v/v) NI-I4HCO3,0.03 M, pH 7.6 Phosphate-buffered saline, pH 7.5 Tris • HC1, 0.01 M, pH 7.5 Tris • HCI, 0.01 M and 1.0 M NaC1, pH 7.5 Na2HPO4/NaPO4,0.1 M, pH 7.2 NaCI, 0.0375 M Experimental Procedures Partially purified human leukocyte interferon preparation, containing major protein contaminants at about 67,000 daltons and 25,000 daltons (Fig. 1), is diluted 1:2 (v/v) with phosphate-buffered saline and then mixed with an equal volume of periodate buffer (0.02 M NaIO4 in 0. l M sodium acetate, pH 4.5). The mixture is vortexed, and the reaction mixture is stored in the dark at 4 ° for 2-4 hr. After the desired period of treatment the mixture is centrifuged at 13,000 g for l0 rain. The precipitate formed during the reaction contains most of the 25,000 dalton protein bul little of the interferon activity. The supernatant fluid, which contains the 67,000 dalton contaminant and most of the interferon activity, is decanted and diluted 1 : 10 (v/v) with 50% ethylene glycol in water to stop the reaction. The preparation is then dialyzed extensively against 0.03 34 NI-I4HCO3 buffer (pH 7.6), and lyophilized. The lyophilized sample is resuspended in a minimum volume of Tris buffer (0.01 M, pH 7.5), and applied to a Sephadex G-100 column (2.6 cm in diameter by 170 cm in length) equilibrated with the same buffer. Samples are eluted with Tris buffer at a flow rate of 1 ml/min, and 10-ml fractions are collected with an LKB Ultrorac Model 7000 fraction collector. Protein concentrations are monitored with a Pharmacia UV I monitor operating at 280 nm with 0.05 to 0.1 absorbance unit full scale (AUFS). Interferon activities are determined by the cytopathic effect inhibition assay2 Fractions containing peak interferon activities well separated from the 67,000 dalton protein contaminant are pooled and applied to a poly(U)8 S. Udenfriend, S. Stein, P. BOhlen, W. Dairman, W. Leimgruber, and M. Weigele, Scie n c e 178, 871 (1972). 9 W. E. Stewart II, "The Interferon System." Springer-Verlag, Vienna and New York, 1979.
484
[69]
PURIFICATION AND CHARACTERIZATION
mol. wt. column marke,,r,S e!uent
.
104 ppt.
"
PIF.
,
l x 1 0 s Interferon units per sample FIG. I. One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of human leukocyte interferon demonstrating the selective removal of the 25,000-dalton noninterferon protein by periodate precipitation and the separation of the 67,000-dalton contaminant protein by gel filtration. Left-hand track represents the protein
[69]
TWO-DIMENSIONAL GEL PURIFICATION
485
Sepharose column (with a bed dimension of 1.6 cm in diameter by 1.5 cm height) equilibrated with the same Tris buffer. Protein concentration is monitored with a Pharmacia UV I monitor operating at 280 nm with 0.01 AUFS. After the samples are applied onto the poly(U)-Sepharose, the column is washed with the same Tris buffer until no detectable protein is further eluted from the column. The column is then eluted with 0.01 M Tris-l.0 M NaCI, pH 7.5. Four-milliliter fractions are collected with a Gilson microfractionator. Fractions are assayed for interferon activities and then pooled, dialyzed against 0.03 M NH4HCO3 buffer, and then lyophilized for application to isoelectric focusing gels.
Two-Dimensional Gel Electrophoresis A modified O'Farrcll two-dimensional gel clectrophoresis system is used to further purify the human leukocyte interferon preparations. The modifications are (a) the omission of reducing agents, such as/3-mcrcaptoethanol, in all solutions; and (b), the increased length of thc isoelectric focusing gel from l I c m to 24 c m with the corresponding increase of the second-dimension S D S gel slab to 35 c m wide by 30 c m in height. Isoclectric focusing is carried out in long capillary gels (2.8 m m in diameter by 24 c m in length). The gels contain 9 M urea, 4 % acrylamide, 0.2% bisacrylamide, 2 % NP-40 (w/v) and 2 % Ampholine (v/v) (LKB, 4: l mixture ofpH 5-7 and p H 3-10). Polymerization is initiatedby addition of T E M E D (7 tzl/10 ml) and a m m o n i u m persulfatc (I txg/10 ml). The gel solution is introduced into the gel tubes with capillary tubing connected via a hypodermic needle to a syringe. The filledgel tubes arc further degassed to assure uniformity. The gels were then processed and prefocused as described by O'Farcll.6 Interferon samples are solubilized in a minimum volume of focusing sample buffer, which contains 9.5 M urea, 2 % (w/v) NP-40 and 2 % A m pholine mixture to a finalprotein concentration of approximately 1/zg//zl; 25-35/zl of solubilized interfcron sample is applied to each isoelcctricfocusing gel. Focusing is carried out at 400 V constant voltage until a minim u m current is obtained. The focusing voltage is then raised to 800 V for markers; each of the other tracks in the slab gel was loaded with approximately the same total units of interferon activity: PIF, partially purified human leukocyte interferon with a specific activity of about 108units per milligramof protein (starting material); IO~ ppt, precipitate formed after periodate treatment, composed of essentially the 25,000-dalton contaminant and very little interferon activity; IO~ sup, supernatant material after periodate treatment, containing most of the interferon activity and the 67,000-dalton contaminant, but devoid of the 25,000dalton species; Sephadex G-100 column eluent: fractions containingpeak interferon activity arefree of the bulk of both the 67,000-dalton and 25,000-dalton contaminant proteins.
486
[69]
PURIFICATION AND CHARACTERIZATION Isoetectric M48,"10
w
a
Focusing pH4.03
•
•
eTK
e
~) 45K
Q
o 30K
m
h 20cm
~
m 14AK 24cm
FIG. 2. Two-dimensional gel electrophoresis of human leukocyte interferon. A two-dimensional polyacrylamide slab of the periodate-precipitated, Sephadex G-100-separated, and poly(U)-Sepharose-chromatographed human leukocyte interferon was stained for protein with Coomassie Brilliant Blue. The two spots of interferon antiviral activities determined by assaying fractions from a duplicate unstained gel correspond to the two stained spots (enclosed area is enlarged and inserted at upper right). The spot on the acidic side corresponds to the interferon species, which has nearly equal antiviral activities on both human and bovine cells, whereas the spot on the basic side corresponds to the interferon species with predominant antiviral activities on bovine ceils.
60 rain before termination. Gels are extruded onto sheets of parafilm with a gel extruder (LAC Enterprise). Gels are immediately frozen at - 70 ° and fractionated into 2.2-ram segments, and each segment is placed in 1 ml of 1% SDS and 0.15 M NaC1 solution and stored for elution and assay. The pH gradient is determined by eluting 1-cm fractions of a gel in 1 ml of 0.0375 M NaC1 overnight and then measuring the pH of the solution at room temperature. Electrophoresis in the second dimension is carded out on slab gels (35 cm × 30 cm x 0.15 cm) with single 30-cm slot. The gel contains 0.45 M urea, 10% acrylamide, 0.8% bisacrylamide, 0.1 M phosphate buffer, pH 7.2, 0.1% SDS. Polymerization is initiated by addition of TEMED (6/zl/10 ml) and ammonium persulfate (0.8 mg/ml). Isoelectric focusing gels are embedded onto the SDS gel slab by molten agarose solution (1% agarose, 2% SDS in 0.1 M sodium phosphate, pH 7.2). The upper buffer is 2% SDS in 0.1 M sodium phosphate, pH 7.2, and the lower buffer is 0.1% SDS in 0.1 M sodium phosphate, pH 7.2. Electrophoresis is at 15 W constant power toward the anode. After electrophoresis for 20 rain,
[70]
ANTIBODY AFFINITY CHROMATOGRAPHY
487
the top electrode buffer is removed and replaced with I ml of 0.1% SDS in 0.1 M sodium phosphate buffer, pH 7.5. Electrophoresis is terminated when the tracking dye bromophenol blue has migrated about 23 cm through the gel. The slab gel is removed and either stained for protein with Coomassie Brilliant Blue or the appropriate area is cut into fractions of 0.5-cm squares and eluted with 1 ml of 1% SDS in 0.15 M NaCI and assayed for interferon activity on both human and bovine cell cultures. 1° This method of two-dimensional gel electrophoresis of periodatetreated preparations can be used to isolate two distinct species of human leukocyte interferon molecules differing in human to bovine activity ratios (Fig. 2). The extra gel lengths employed in both the isoelectric focusing dimension and SDS-PAGE dimension is necessary to resolve completely the various species of human leukocyte interferon. l0 L. S. Lin, M. Wiranowska-Stewart, T. Chudzio, and W. E. Stewart II, J. Gen. Virol. 39, 125 (1978).
[70] A n t i b o d y By
Affinity Chromatography Leukocyte Interferon
of Human
K U R T BERG a n d IVER HERON
In theory, the antibody affinity chromatography technique is very simple for obtaining pure human leukocyte interferon proteins, provided that only strictly monospecific anti-interferon immunoglobulins are used and no fortuitous (nonspecific) immunological crossing-over occurs. Accordingly, the immunoglobulins are immobilized by covalently binding to a suitable matrix, for example, Sepharose 4B, without destroying or distorting the antigenic sites, and a small column is prepared. At neutral pH (pH 6.5-7.5) the immobilized antibodies will bind only the interferon proteins when a crude preparation of human leukocyte interferon is passed through the column. After a thorough wash the interferon is released by disrupting the antigen-antibody complex in a suitable manner, such as by lowering the pH (to 2.4), and pure interferon proteins are obtained with a high recovery (75-100%). The term "monospecific antibodies" implies, in this context, that the anti-interferon immunoglobulin preparation reacts only with the interferon proteins, n o t with any of the contaminating proteins present in an overwhelming amount in crude interferon preparations. 1'2 To compeni K. Berg, Scand. J. lmmunol. 6, 77 (1977). z K. Berg, I. Heron, and R. Hamilton, Stand. J. lmmunol. 8, 429 (1978).
METHODS IN ENZYMOLOGY, VOL. 78
Copyright © 1981 by Academic Press, Inc. All fights of reproduction in any form reserved. ISBN 0-12-181978-7