- Email: [email protected]
[71] Partial purification of human leukocyte interferon on a large scale
[71]
PURIFICATION OF HUMAN LEUKOCYTE INTERFERON
499
units per milligram of proteins) is only approximate, since the protein estimate involves comparison of the intensity of the stain between the interferon proteins and protein markers. Accordingly, the specific activity given as 2 × 109 units/rag in Table I is only a rough estimate. Possible differential loss of proteins during staining a n d / o r differential staining ability of the interferon proteins (or markers) could produce substantial variation. For further details, the reader is referred to Berg and Heron. "~'tl Acknowledgments This investigation was supported by the Danish Cancer Society. The excellent technical assistance provided by Kaj Vesterg~trdand Karin Durup is gratefully acknowledged, " K. Berg and I. Heron, J. Gen. Virol. 50, 44l (1980).
[71] Partial Purification of Human Leukocyte on a Large Scale By
Interferon
KARl CANTELL, SINIKKA HIRVONEN, and VESA KOISTINEN
The method worked out in our laboratories for the large-scale purification of human leukocyte interferon 1-3 has been modified further. The table shows the purification scheme. Crude interferon is pooled in three 7.8-liter batches in round 10-liter flasks and stored overnight at 4 °. The three batches are successively precipitated in 0.5 M KSCN at room temperature. A high-temperature combined electrode (465-35-90-K9, Dr. W. Ingold AG, CH-8092, ZOrich) is rinsed with 70% ethanol, exposed to ultraviolet irradiation, and submerged into the interferon solution for continuous monitoring of the pH with a PHM84 research pH meter (Radiometer, Copenhagen). Throughout the purification process, all changes in the pH are made very slowly while the solutions are stirred briskly. KSCN precipitation is performed at pH 3.8, and the precipitate is centrifuged at 4 °"
For each batch 94% ethanol, to which no acid is added, is premeasured and kept at - 2 0 °. The containers of the Waring blender are also kept at - 2 0 °. Ethanol extraction is carried out on a horizontal laminarK. Cantell, S. Hirvonen, K. E. Mogensen, and L. Pyh~il~, in "The Production and Use of Interferon for the Treatment and Prevention of Human Virus Infections" (C. Waymouth, ed.), p. 35. Tissue Culture Association, Rockville, Maryland, 1974. 2 K. Cantell and S. Hirvonen, Tex. Rep. Biol. Med. 35, 138 (1977). K. Cantell and S. Hirvonen, J. Gen. Virol. 39, 541 (1978).
METHODS IN ENZYMOLOGY, VOL. 78
Copyright © 1981 by Academic Press, Inc. All fights of reproduction in any form reserved. 1SBN 0-12-181978-7
500
[71]
PURIFICATION A N D CHARACTERIZATION
7
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[71]
PURIFICATION OF HUMAN LEUKOCYTE INTERFERON
6 :ff Z t)
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502
PURIFICATION A N D CHARACTERIZATION
[71]
flow bench at room temperature. The KSCN supernatant is discarded, and cold ethanol is poured on the pellet, which is shaken loose and transferred to the container of the blender. The centrifuge bottles are rinsed with cold ethanol. The suspension is blended at 21,000 rpm four times, each time for 5 sec at 5-sec intervals. The ethanol extracts are then centrifuged at about - 5 °, and the supernatants are pooled in a 5-liter glass bottle. The pH of the supernatants is about 5.1, and the temperature is kept between 0 and 4 ° with the aid of ice water. The pH is raised to 5.5 by the dropwise addition of 0.1 N NaOH. This takes about 1 hr. After centrifugation the precipitate is removed and the pH of the supernatant is raised to 5.75. The precipitate is again discarded after centrifugation, and the pH of the supernatant is raised to 8.0. After centrifugation, the pH 8.0 precipitate is dissolved as follows: the supernatants are decanted and the l-liter polypropylene centrifuge bottles are placed immediately on crushed ice. About 90 g of cold sterile glass beads (diameter 4 - 5 mm) and cold 0.1 M phosphate buffer, pH 8.0, containing 0.5 M KSCN are added. The sediment is shaken loose by strong rotating movements, and the suspension is agitated slowly overnight on a magnetic stirrer at 4 °. The suspensions are pooled, and about 10 ml of the aforementioned buffer is used to rinse each bottle and its glass beads. The suspension is stored at - 7 0 °. It can be stored for at least a year with no loss of interferon activity. Two batches, concentrated 50-fold and partially purified as described above, are routinely pooled for further concentration and purification. The suspensions are thawed in cold water. As soon as the suspension is partly thawed, it is agitated on a magnetic stirrer for 3 hr at 4°. Insoluble material is removed by centrifugation at 1600 g for 30 min at 4 °. The cold interferon is then precipitated at room temperature. By addition of 2 N HC1 the pH is slowly lowered to 5.30-5.35 when the first visible precipitate appears. The suspension is stirred for 10 min while precipitation continues with no further addition of HC1. By addition of 1 N HCI the pH is lowered very slowly to 5.1 or to 4.7. The suspension is stirred for 20 min and centrifuged as above in 800-ml round-bottom metal cups (M. Christ, 3360 Osterode, FRG). The pH of the supernatant is lowered to 2.8 by addition of 2 N HCI. The suspension is stirred for 10 min and centrifuged as above. The sediments are dissolved by first placing a magnetic bar (9 x 20 mm) directly onto the sediment which, agitated on a magnetic stirrer, is homogenized into a "porridge" on a laminar-flow bench at room temperature. Two solutions, (a) 0.1 M phosphate buffer, pH 8.0, containing 0.5 M KSCN and ~ volume of 1 N NaOH, and (b) the same solution without
[71]
PURIFICATION OF HUMAN LEUKOCYTE INTERFERON
503
FIG. 1. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of purified human leukocyte interferons A and B. The electrophoresis was performed in 14-cmgel slabs containing 12% acrylamide. The buffer system was that described by U. K. Laemmli [Nature (London) 227, 680 (1970)]. Samples were prepared for electrophoresis by heating in the presence of 2% SDS for 1 min at 100°. After electrophoresis, gels were stained with Coomassie Brilliant Blue. On both sides of the interferon preparations are standard proteins of known molecular weights. From the top they are (molecular weights in parentheses): phosphorylase b (94,000), bovine serum albumin (67,000), ovalbumin (43,000), carbonic anhydrase (30,000), soybean trypsin inhibitor (20d00), and lactalbumin (14,400).
50 4
PURIFICATION AND CHARACTERIZATION
[71]
NaOH are then alternately added dropwise. The pH is checked with indicator paper while the sediments are slowly dissolving. The pH should not rise above 8.0. P-IF A dissolves more readily than P-IF B; the former dissolves around pH 7, and the latter between 7.5 and 8. The P-IF B preparations are routinely dissolved in 1/500th, and the P-IF A preparations in 1/1000th, of the original volume of crude interferon. The suspensions are kept for 3 hr on a magnetic stirrer at room temperature. They are then transferred into sterilized dialysis bags (Arthur H. Thomas Co. Philadelphia, Pennsylvania, Cat. No 3787-D32) and dialyzed for 2 - 3 hr against 100 volumes of PBS at room temperature. The dialysis is continued overnight and for an additional day against 100 volumes of fresh PBS at 4 °. During the dialysis some noninterferon precipitate forms in P-IF B and a little in P-IF A. The dialysis bags are wiped with 70% ethanol and cut with sterile scissors; their contents are harvested with pipettes. The interferons are centrifuged, 31,500 g for 60 min, and the supernatants are stored at - 7 0 °. P-IF B preparations are brownish; P-IF A preparations have less color. The total recovery of P-IF A and P-IF B is about 60%. The relative recovery of the two preparations depends on the pH at which P-IF B is removed. The lower the pH, the higher the proportion of P-IF B. When the precipitation is done at pH 5.1, about 25% of the recovered P-IF is of type B and 75% of type A. When the pH is 4.7, the proportions of P-IF A and P-IF B are reversed. The specific activities of P-IF A and P-IF B range between 0.8 and 7 x 106 IU per milligram of protein. Figure 1 shows the striking dissimilarity of the impurities in P-IF A and P-IF B. Albumin is a prominent impurity in P-IF A. The main contaminant of P-IF B is a hydrophobic protein with a molecular weight of about 28,000. Both P-IF A and P-IF B are heterogeneous, but the latter contains a higher proportion of small molecular forms (Fig. 2). Note Added in Proof We have lowered the concentration of agamma serum in the production medium from 2.4 to 1.8 mg of protein per milliliter. This has not essentially changed the titers of crude interferon. The purification procedure has been modified as follows: The precipitations of the ethanol supernatants are done at 0°C in a refrigerated bath (EK12 and EK3, Haake, Berlin-Karlsruhe). The pH 8.0 precipitate is dissolved on an orbital shaker (Bellco Glass Inc., Vineland, N J) for 2 hr and kept overnight at 4 °. The precipitations at P-IF A and P-IF B are done at 4 °. The first visible precipitate appears at pH 5.20-5.30. We have produced and purified 470 liters of crude interferon after the
[72]
RECOMBINANT LEUKOCYTE
IF
30K- ~ ~ . ~ _
PURIFICATION
--
P-IFB j
505
20K
2oK
P'IFA
30K 20K
~
~
I
20K
1OK
Migration
Fro. 2. Size distribution of interferon molecules in purified human leukocyte interferons A and B (P-1F A and P-IF B). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed as described in Fig. 1. For locating interferon activity, the nonstained gels were cut into 2-mm slices that were eluted for 2 days at 37° with PBS, pH 7.4, containing 0.5% SDS and 2% calf serum. The regions of the gels containing the standards were stained after the sample slices had been removed, c.anh. = carbonic anhydrase; tr.i. = soybean trypsin inhibitor; la. = a-lactalbumin. adoption o f these modifications. The total recovery of P-IF A and P-IF B has been about 90% and the specific activities have ranged between 2.5 × 106 and 1.25 × 107 I U per milligram o f protein.
[72] Purification of Recombinant Human Leukocyte Interferon (IFLrA) with Monoclonal Antibodies
By
THEOPHIL
STAEHELIN,
DONNA
S.
HOBBS,
HSlANG-FU KUNG, and S~DNEV PESTKA We have isolated and identified D N A recombinants containing sequences for h u m a n leukocyte and fibroblast interferons. 1 One such clone was used to isolate a full-length e D N A recombinant that was reconS. Maeda, R. McCandliss, M. Gross, A. Sloma, P. C. Familletti, J. M. Tabor, M. Evinger, W. P. Levy, and S. Pestka, Proc. Natl. Acad. Sci. U.S.A. 77, 7010 (1980).
METHODS IN ENZYMOLOGY, VOL. 78
Copyright~ 1981by AcademicPress, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181978-7
PURIFICATION OF HUMAN LEUKOCYTE INTERFERON
499
units per milligram of proteins) is only approximate, since the protein estimate involves comparison of the intensity of the stain between the interferon proteins and protein markers. Accordingly, the specific activity given as 2 × 109 units/rag in Table I is only a rough estimate. Possible differential loss of proteins during staining a n d / o r differential staining ability of the interferon proteins (or markers) could produce substantial variation. For further details, the reader is referred to Berg and Heron. "~'tl Acknowledgments This investigation was supported by the Danish Cancer Society. The excellent technical assistance provided by Kaj Vesterg~trdand Karin Durup is gratefully acknowledged, " K. Berg and I. Heron, J. Gen. Virol. 50, 44l (1980).
[71] Partial Purification of Human Leukocyte on a Large Scale By
Interferon
KARl CANTELL, SINIKKA HIRVONEN, and VESA KOISTINEN
The method worked out in our laboratories for the large-scale purification of human leukocyte interferon 1-3 has been modified further. The table shows the purification scheme. Crude interferon is pooled in three 7.8-liter batches in round 10-liter flasks and stored overnight at 4 °. The three batches are successively precipitated in 0.5 M KSCN at room temperature. A high-temperature combined electrode (465-35-90-K9, Dr. W. Ingold AG, CH-8092, ZOrich) is rinsed with 70% ethanol, exposed to ultraviolet irradiation, and submerged into the interferon solution for continuous monitoring of the pH with a PHM84 research pH meter (Radiometer, Copenhagen). Throughout the purification process, all changes in the pH are made very slowly while the solutions are stirred briskly. KSCN precipitation is performed at pH 3.8, and the precipitate is centrifuged at 4 °"
For each batch 94% ethanol, to which no acid is added, is premeasured and kept at - 2 0 °. The containers of the Waring blender are also kept at - 2 0 °. Ethanol extraction is carried out on a horizontal laminarK. Cantell, S. Hirvonen, K. E. Mogensen, and L. Pyh~il~, in "The Production and Use of Interferon for the Treatment and Prevention of Human Virus Infections" (C. Waymouth, ed.), p. 35. Tissue Culture Association, Rockville, Maryland, 1974. 2 K. Cantell and S. Hirvonen, Tex. Rep. Biol. Med. 35, 138 (1977). K. Cantell and S. Hirvonen, J. Gen. Virol. 39, 541 (1978).
METHODS IN ENZYMOLOGY, VOL. 78
Copyright © 1981 by Academic Press, Inc. All fights of reproduction in any form reserved. 1SBN 0-12-181978-7
500
[71]
PURIFICATION A N D CHARACTERIZATION
7
;h V"
z
g/ >.
u~ Z .¢
:Z
gfl 0
a: C~
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,~O e~ O
...q e~
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~o =
,~=
[71]
PURIFICATION OF HUMAN LEUKOCYTE INTERFERON
6 :ff Z t)
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t::l
-61-:.
kl
[.-
,-2
-6~.
H
-6 r/l
oo
501
502
PURIFICATION A N D CHARACTERIZATION
[71]
flow bench at room temperature. The KSCN supernatant is discarded, and cold ethanol is poured on the pellet, which is shaken loose and transferred to the container of the blender. The centrifuge bottles are rinsed with cold ethanol. The suspension is blended at 21,000 rpm four times, each time for 5 sec at 5-sec intervals. The ethanol extracts are then centrifuged at about - 5 °, and the supernatants are pooled in a 5-liter glass bottle. The pH of the supernatants is about 5.1, and the temperature is kept between 0 and 4 ° with the aid of ice water. The pH is raised to 5.5 by the dropwise addition of 0.1 N NaOH. This takes about 1 hr. After centrifugation the precipitate is removed and the pH of the supernatant is raised to 5.75. The precipitate is again discarded after centrifugation, and the pH of the supernatant is raised to 8.0. After centrifugation, the pH 8.0 precipitate is dissolved as follows: the supernatants are decanted and the l-liter polypropylene centrifuge bottles are placed immediately on crushed ice. About 90 g of cold sterile glass beads (diameter 4 - 5 mm) and cold 0.1 M phosphate buffer, pH 8.0, containing 0.5 M KSCN are added. The sediment is shaken loose by strong rotating movements, and the suspension is agitated slowly overnight on a magnetic stirrer at 4 °. The suspensions are pooled, and about 10 ml of the aforementioned buffer is used to rinse each bottle and its glass beads. The suspension is stored at - 7 0 °. It can be stored for at least a year with no loss of interferon activity. Two batches, concentrated 50-fold and partially purified as described above, are routinely pooled for further concentration and purification. The suspensions are thawed in cold water. As soon as the suspension is partly thawed, it is agitated on a magnetic stirrer for 3 hr at 4°. Insoluble material is removed by centrifugation at 1600 g for 30 min at 4 °. The cold interferon is then precipitated at room temperature. By addition of 2 N HC1 the pH is slowly lowered to 5.30-5.35 when the first visible precipitate appears. The suspension is stirred for 10 min while precipitation continues with no further addition of HC1. By addition of 1 N HCI the pH is lowered very slowly to 5.1 or to 4.7. The suspension is stirred for 20 min and centrifuged as above in 800-ml round-bottom metal cups (M. Christ, 3360 Osterode, FRG). The pH of the supernatant is lowered to 2.8 by addition of 2 N HCI. The suspension is stirred for 10 min and centrifuged as above. The sediments are dissolved by first placing a magnetic bar (9 x 20 mm) directly onto the sediment which, agitated on a magnetic stirrer, is homogenized into a "porridge" on a laminar-flow bench at room temperature. Two solutions, (a) 0.1 M phosphate buffer, pH 8.0, containing 0.5 M KSCN and ~ volume of 1 N NaOH, and (b) the same solution without
[71]
PURIFICATION OF HUMAN LEUKOCYTE INTERFERON
503
FIG. 1. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of purified human leukocyte interferons A and B. The electrophoresis was performed in 14-cmgel slabs containing 12% acrylamide. The buffer system was that described by U. K. Laemmli [Nature (London) 227, 680 (1970)]. Samples were prepared for electrophoresis by heating in the presence of 2% SDS for 1 min at 100°. After electrophoresis, gels were stained with Coomassie Brilliant Blue. On both sides of the interferon preparations are standard proteins of known molecular weights. From the top they are (molecular weights in parentheses): phosphorylase b (94,000), bovine serum albumin (67,000), ovalbumin (43,000), carbonic anhydrase (30,000), soybean trypsin inhibitor (20d00), and lactalbumin (14,400).
50 4
PURIFICATION AND CHARACTERIZATION
[71]
NaOH are then alternately added dropwise. The pH is checked with indicator paper while the sediments are slowly dissolving. The pH should not rise above 8.0. P-IF A dissolves more readily than P-IF B; the former dissolves around pH 7, and the latter between 7.5 and 8. The P-IF B preparations are routinely dissolved in 1/500th, and the P-IF A preparations in 1/1000th, of the original volume of crude interferon. The suspensions are kept for 3 hr on a magnetic stirrer at room temperature. They are then transferred into sterilized dialysis bags (Arthur H. Thomas Co. Philadelphia, Pennsylvania, Cat. No 3787-D32) and dialyzed for 2 - 3 hr against 100 volumes of PBS at room temperature. The dialysis is continued overnight and for an additional day against 100 volumes of fresh PBS at 4 °. During the dialysis some noninterferon precipitate forms in P-IF B and a little in P-IF A. The dialysis bags are wiped with 70% ethanol and cut with sterile scissors; their contents are harvested with pipettes. The interferons are centrifuged, 31,500 g for 60 min, and the supernatants are stored at - 7 0 °. P-IF B preparations are brownish; P-IF A preparations have less color. The total recovery of P-IF A and P-IF B is about 60%. The relative recovery of the two preparations depends on the pH at which P-IF B is removed. The lower the pH, the higher the proportion of P-IF B. When the precipitation is done at pH 5.1, about 25% of the recovered P-IF is of type B and 75% of type A. When the pH is 4.7, the proportions of P-IF A and P-IF B are reversed. The specific activities of P-IF A and P-IF B range between 0.8 and 7 x 106 IU per milligram of protein. Figure 1 shows the striking dissimilarity of the impurities in P-IF A and P-IF B. Albumin is a prominent impurity in P-IF A. The main contaminant of P-IF B is a hydrophobic protein with a molecular weight of about 28,000. Both P-IF A and P-IF B are heterogeneous, but the latter contains a higher proportion of small molecular forms (Fig. 2). Note Added in Proof We have lowered the concentration of agamma serum in the production medium from 2.4 to 1.8 mg of protein per milliliter. This has not essentially changed the titers of crude interferon. The purification procedure has been modified as follows: The precipitations of the ethanol supernatants are done at 0°C in a refrigerated bath (EK12 and EK3, Haake, Berlin-Karlsruhe). The pH 8.0 precipitate is dissolved on an orbital shaker (Bellco Glass Inc., Vineland, N J) for 2 hr and kept overnight at 4 °. The precipitations at P-IF A and P-IF B are done at 4 °. The first visible precipitate appears at pH 5.20-5.30. We have produced and purified 470 liters of crude interferon after the
[72]
RECOMBINANT LEUKOCYTE
IF
30K- ~ ~ . ~ _
PURIFICATION
--
P-IFB j
505
20K
2oK
P'IFA
30K 20K
~
~
I
20K
1OK
Migration
Fro. 2. Size distribution of interferon molecules in purified human leukocyte interferons A and B (P-1F A and P-IF B). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed as described in Fig. 1. For locating interferon activity, the nonstained gels were cut into 2-mm slices that were eluted for 2 days at 37° with PBS, pH 7.4, containing 0.5% SDS and 2% calf serum. The regions of the gels containing the standards were stained after the sample slices had been removed, c.anh. = carbonic anhydrase; tr.i. = soybean trypsin inhibitor; la. = a-lactalbumin. adoption o f these modifications. The total recovery of P-IF A and P-IF B has been about 90% and the specific activities have ranged between 2.5 × 106 and 1.25 × 107 I U per milligram o f protein.
[72] Purification of Recombinant Human Leukocyte Interferon (IFLrA) with Monoclonal Antibodies
By
THEOPHIL
STAEHELIN,
DONNA
S.
HOBBS,
HSlANG-FU KUNG, and S~DNEV PESTKA We have isolated and identified D N A recombinants containing sequences for h u m a n leukocyte and fibroblast interferons. 1 One such clone was used to isolate a full-length e D N A recombinant that was reconS. Maeda, R. McCandliss, M. Gross, A. Sloma, P. C. Familletti, J. M. Tabor, M. Evinger, W. P. Levy, and S. Pestka, Proc. Natl. Acad. Sci. U.S.A. 77, 7010 (1980).
METHODS IN ENZYMOLOGY, VOL. 78
Copyright~ 1981by AcademicPress, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181978-7