- Email: [email protected]
[24] Purification of recombinant human IFN-α2
166
PURIFICATION OF INTERFERONS
[24]
[24] P u r i f i c a t i o n o f R e c o m b i n a n t H u m a n I F N - a 2 By DAVID R. THATCHER and NIKOS PANAYOTATOS
Alpha or leukocyte interferon represents a homologous family of proteins with antiviral activity secreted by nearly all virus-infected human cells. ~ Because in v i v o levels of synthesis are extremely low, interferon purification from natural sources has been a challenging technical problem. 2 Large volumes of either peripheral blood leukocyte sl-3 (buffy coats) or immortalized cell lines 4-~° are required in conjunction with sophisticated separation techniques such as reversed phase HPLC 2,J~ or monoclonal immunoaffinity chromatography. ~2,j3 The advent of gene technology has afforded a more efficient and cost effective approach to production of interferon. Microorganisms, expressing individual interferon genes at high level, 2 now provide an abundant source of protein for purification. As described in this chapter, purification of recombinant interferon can be achieved by standard methodology and can easily be scaled up to meet the precise and reproducible criteria required for manufacturing. In fact, human IFN-c~2 has been purified and crystallized on a commercial scale by Nagabushan and co-workers. J4 The Hu-IFN-a2 and the closely related Hu-IFN-c~A genes were isolated, respectively, by Nagata e t al. j5 and Streuli e t a l . j6 o n the one hand and by Maeda e t a/. ~7and Goeddel e t al. 18 on the other. The cDNA clones S. Pestka and S. Baron, this series, Vol. 78, p. 3. 2 S. Pestka, Arch. Biochem. Biophys. 221, I (1983). 3 K. Berg, C. A. Ogburn, K. Paucker, K. E. Mogensen, and K. Cantell, J. lmmunol. 114, 640 (1975). 4 H. Strander, K. E. Mogensen, and K. Cantell, J. C/in. Mierobiol. 1, 116 (1975). 5 A. Mizrahi, this series, Vol. 78, p. 54. 6 G. Bodo, this series, Vol. 78, p. 69. 7 F. Klein and R. T. Ricketts, this series, Vol. 78, p. 75. 8 p. C. Familletti, L. Costello, C. A. Rose, and S. Pestka, this series, Vol. 78, p. 83. 9 T. M. Powledge, Bio/Technology 214 (1984). to A. W. Phillips, N. B. Finter, C. J. Burman, and G. D. Ball, this volume [4]. H M. Rubinstein, S. Rubinstein, P. C. Familletti, R. S. Miller, A. A. Waldman, and S. Pestka, Proe. Natl. Aead. Sci. U.S.A. 76, 640 (1979). iz D. Secher and D. C. Burke, Nature (London) 285, 446 (1980). ~3 T. Staehelin, D. S. Hobbs, H.-F. Kung, and S. Pestka, this series, Vol. 78, p. 505. 14 T. L. Nagabushan and P. Leibowitz, in "Interferon alpha 2: preclinical and clinical evaluation," pp. 1-12. Nijhoff, The Hague, 1985. t5 S. Nagata, H. Taira, A. Hall, L. J o h n s r u d , M. Streuli, J. Escodi, W. Boll, K. Cantell, and C. W e i s s m a n n , Nature (London) 284, 316 (1980).
M E T H O D S IN E N Z Y M O L O G Y , VOL. 119
Copyright © 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.
[24]
PURIFICATION OF HUMANIFN-a2
~x~o2O's~
V
167
Cene
pNKS97J
Ori ~> FIO. 1. Structure of the pl.TT.a2.copl expression vector, pI.TT.c~2.cop! consists of the pBR322 sequence between the unique SalI (position 650) and EcoRl (position 4359) restriction sites and 112 bp of bacteriophage T7 DNA containing the ribosome binding site for gene 1.1.19 The promoter pl described in this work introduced at the EcoRI site and the human leukocyte IFN-a2 gene at the Sail site. The positions of the unique PstI and AvaI restriction sites are shown. The origin and direction of replication, the positions of the primer RNA (Pm) and the RNA I (pl) promoters, as well as the copl point mutation at position 3029 of the pBR322 map are also indicated.
were prepared from mRNA derived from human leukocytes induced with Sendai virus. The region coding for the natural interferon signal peptide (which cannot be processed correctly by Escherichia coli) was removed, and the gene sequence corresponding to the extracellular protein was expressed in E. coli. ~8 The Plasmid Vector
A plasmid containing the IFN-a2 gene was kindly provided by C. Weissmann. A 720 bp H i n d I I I - P s t I fragment was digested with S 1 nuclease to provide blunt ends and was inserted into plasmid pNKS97 at a unique SalI site rendered blunt with SI nuclease (Fig. 1). Plasmid
ii, M. Slreuli, S. Nagata, and C. Weissmann, Science 209, 1343 (1980). ~7 S. Maeda, R. McCandliss, M. Gross, A. Sloma, P. C. Familletli, J. M. Tabor, M. Evinger, W. P. Levy, and S. Pestka, Proc. Natl. Acad. Sci. U.S.A. 77, 7010 (1980); 78, 4648 (1981). 's D. V. Goeddel, E. Yelverton, A. UIIrich, H. L. Heyneker, G. Miozzari, W. Holmes, P. H. Seeburg, T. Dull, L. May, N. Slebbing, R. Crea, S. Maeda, R. McCandliss, A. Sloma, J. M. Tabor, M. Gross, P. C. Familletti, and S. Peslka, Nature (London) 287, 411 (1980).
168
PURIFICATION OF INTERFERONS
[24]
pNKS97 is a versatile vector for gene expression in E. coli. 19This plasmid consists of 3709 bp of the pBR322 sequence between the SalI and EcoRI sites containing the/3-1actamase (amp R) and replication regions as well as a 112 bp fragment from bacteriophage T7 which contains the ribosome binding site for gene 1.1 (Fig. 1). A unique EcoRI site allows the insertion of promoter fragments upstream from the ribosome binding site whereas the unique SalI site permits the insertion of the gene to be expressed (in this case the IFN-a2 gene) in phase with an initiating methionine codon. The promoter used was the PI promoter from ColE1 which promotes the transcription of a small RNA molecule (RNAI) which is involved in the control of plasmid replication. A copy number mutation of this plasmid [pI.T7.a2.copl] was isolated by selection on L-broth agar plates containing 20 mg/ml methicillin. 2° Fermentation The bacteria harboring the pI.T7.a2.copl plasmid were maintained on L-agar plates containing l0 mg/ml ampicillin and were subcultured at weekly intervals. Cultures were allowed to grow at 37 ° for 20 hr into stationary phase in overfilled 31 shake flasks containing 1.6 liter of standard L-broth at low agitation. IFN-a2 expression was of the order of 515 × 109 units/liter/OD590 (16-47 mg/liter), or 10-30% of total cellular protein. Purification of IFN-a2 Increased levels of interferon expression in this system lead to a proportionately increasing fraction of interferon protein laid down as an intracellular insoluble aggregate (Fig. 2). At high levels of expression, over 95% of the interferon synthesized by the pI.T7.a2.copl system is in an insoluble physical state. This aggregation of foreign or mutant proteins expressed at high levels in E. coli is a well documented phenomenon 21,22 and can be exploited to advantage in protein purification strategies. Upon cell breakage, the soluble cell constituents, which include the majority of the contaminating E. coli proteins, can be washed away by successive 19 N. Panayotatos and K. Truong, Nucleic Acids Res. 9, 5679 (1981). 20 N. Panayotatos, A. Fontaine, and K. Truong, J. Cell. Biochem., Suppl. 7B, Abstr. No. 765 (1983). 2J A. L. Goldberg and A. C. St. John, Annu. Rev. Biochem. 45, 747 (1976). 22 D. C. Williams, R. M. van Frank, W. L. Muth, and J. P. Burnen, Science 215,687 (1982).
[24]
PURIFICATIONOF HUMAN IFN-o~2 10.C
169
o
i
z LL W
_J
0 0
C) .J 0 V)
0 0
W (.9 1.0
0
0 0
0
0
Z ul U W Q_ 00 0
(.9 0 ._J
0.1C 7.0
I
I
8.0
90
L O G SPECIFIC A C T I V I T Y
0
o
I
10.0 {U/GRAM)
FIG. 2. The fraction of soluble intracellular 1FN-c~2 at different levels of e x p r e s s i o n . Total interferon expression (abscissa) was determined by bioassay of cells lysed in 0 . 1 % S D S a n d is expressed in terms of units per gram fresh weight o f E. coli cells. To determine
the fraction of soluble interferon, cells were first sheared with a french press and then centrifuged at 10000 g for 1 hr. The supernatant was then bioassayed and the resulting value expressed as a p e r c e n t a g e o f the total interferon produced.
resuspension and centrifugation steps. Subsequently, the insoluble contaminating membrane proteins of this washed pellet may be selectively solubilized to leave an insoluble pellet whose major proteinaceous constituent is IFN-o~2. The interferon aggregate is then solubilized by a chaotropic solvent and renatured to give a near homogeneous solution of IFN-a2 which can be purified by ion-exchange, metal chelate, and gel permeation chromatography, followed by recrystallization.
Buffers Buffer A: 0. I M Tris. HCI, 0.05 M EDTA, pH 7.5 Buffer B: 2 M guanidine. HC1 treated with activated charcoal and filtered through a 0.22-/~m filter Buffer C: 8 M guanidine. HC1, purified as above Buffer D: 0.04 M sodium dihydrogen phosphate Buffers E and F as D but adjusted to pH 5.5 and 6.0, respectively, with 1 M sodium hydroxide.
170
PURIFICATION OF INTERFERONS
[24]
Cell Breakage The inherent advantage offered by the observed partitioning of IFNa2 into inclusion bodies can only be exploited fully if near total cell breakage is achieved. As the cells are fermented into late stationary phase and are consequently difficult to rupture by mechanical shear, pretreatment with lysozyme has been adopted in order to destroy the integrity of the peptidoglycan layer of the cell wail. This treatment, as well as increasing the efficiency of cell breakage, also reduces the level of carbohydrate in the final washed pellet. The method described below is designed for 100 g to 1 kg of cell pellet. Cells were harvested from fermentation broth by centrifugation at 5000 g. The cells were weighed and placed in a chilled Waring Blendor. A 2-fold (w/v) excess of Buffer A was added, with blending at low speed for 30 sec. Solid sucrose was then dissolved in the cell suspension to give a final concentration of 30% (w/v) followed by the addition of 1 mg of lysozyme hydrochloride for each gram of original cell pellet. After incubation for 1 hr at 30°, the cell suspension was diluted with an equal volume of chilled deionized water. The viscous suspension was thinned by blending with a Kinematica Polytron PT45 and passed once through a french press at a pressure of 800 bar (about 12,000 Ib/infl). The solid components of the suspension, interferon aggregate and cell membrane, were then collected by centrifugation at 10000 g for 30 min. The pellet was resuspended in Buffer B and recentrifuged. This washing procedure was then repeated two more times.
Extraction The final washed pellet was solubilized in a 10-fold (w/v) excess of Buffer C and immediately diluted 4-fold with Buffer A. The suspension was then centrifuged at 10000 g for 30 min and the supernatant dialyzed four times against 20 volumes of distilled water at 4° over a 24-hr period. The cloudy brown precipitate that formed was sedimented at 10000 g for 30 min, the precipitate discarded, and the clear colorless supernatant collected.
Column Chromatography The IFN-o~2 at this stage comprised >90% of the protein in solution as judged by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on samples reduced with 2-mercaptoethanol. Nonreduced samples, however, run on the same SDS-PAGE system, showed a doublet band; the
[24]
PURIFICATIONOF HUMANIFN-a2
171
upper band comigrated with reduced interferon and the lower band with native oxidized interferon. 23 The pH of the solution was reduced to 4.0 by the addition of concentrated orthophosphoric acid and the solution applied to a 5 x 10 cm column of SP Sephadex A-50 equilibrated with Buffer D. The column was then washed with 1 liter of Buffer D followed by 500 ml each of Buffers E and F. Fractions (50 ml) were collected and those containing interferon, as judged by SDS-PAGE, were pooled and passed through a sintered glass funnel containing a bed of DEAE-Sephadex A20 (5 × 10 cm diameter) equilibrated with Buffer F. The flow through from this column was then adjusted to 1 M with respect to NaCI concentration and applied to a copper chelate column. A column (2.5 x 5 cm) of chelating Sepharose 6B (Pharmacia, Uppsala) was prepared as described by the manufacturer. After washing with 9 bed volumes of 1 M NaCI the column was charged with C u 2+ ions 24 by applying a solution of I mg/mi copper sulfate. The column was then repacked with I cm of unchelated matrix at the bottom of the column to mop up any leached copper ion. The column was then washed successively with Buffers F, E, and D all containing 1 M NaCI. Fractions (50 ml) were collected and those containing oxidized interferon as judged by SDS-PAGE run under nonreducing conditions were pooled. The pooled solution was raised to 80% ammonium sulfate saturation by the addition of the solid. The flocculent white precipitate which formed was collected by centrifugation and redissolved in a minimum volume of Buffer F. The concentrate was then applied to a 5 x 100 cm column of Sephacryl S-200 equilibrated with Buffer D containing 1 M NaCI. Fractions (5 ml) were collected and the elution profile monitored at 280 nm. The protein peak eluted as an asymmetric peak with a retention volume corresponding to a molecular weight of approximately 40,000 to 60,000. The major portion of this peak was pooled and the ammonium sulfate saturation raised to 80%. The white precipitate which formed was collected by centrifugation and suspended in a minimum volume of Buffer F. The suspension was then dialyzed against 5 liters of buffer F diluted 100fold. The precipitate disappeared within 30 min and after 48 hr the dialysis bag was filled with microcrystals (Fig. 3). These crystals were collected by low speed centrifugation in a bench centrifuge and stored moist at 4°.
~.3 S. Pestka, B. Kelder, D. K. Tarnowski, and S. J. Tarnowski, Anal. Biochem. 132, 328 (1983). 24 K. C. Chada, this series, Vol. 78. p. 220.
FIG. 3. P h a s e - c o n t r a s t m i c r o s c o p y of I F N - a 2 m i c r o c r y s t a l s . Magnification, x 100. PURIFICATION OF IFN-c~2"
Stage Suspended cells Cell homogenate Supernatant Supernatant of T w e e n w a s h Dialysed guanidine extract Supernatant SP-Sephadex eluate Combined Cu-chelate eluate Combined Sephacryl 5200 fractions Crystallized
Yield (%)
Specific activity (units/mg)
1.6 x 1012
100
2.9 x 106
1.1 × 109
3.7 x 1012
--
9.6 × 107
3140 1080
1.2 x 10s 1.1 x 107
3.7 x 10 H 1.2 × 10 I°
23 0.8
1.5 x 107 5.9 x 106
3160
1.2 x 108
3.7 x 1011
23
--
3060 550
1.2 x 10s 1.1 × 109
3.7 x 10 H 6 x I0 II
23 38
---
500
9.3 x 108
1.4 x 1012
87
--
308
8 x 108
2.4 x 1011
15
--
612
2.7 x lO8
1.7 x lO 11
11
3.2 x 10s
Volume (ml)
Activity/ml (units/ml)
Total (units)
1500
1.1 x l09
3400
A b o u t 770 g of E. coli containing I F N - a 2 was used as the starting material.
[24]
PURIFICATION OF HUMAN 1FN-a2
173
FIG. 4. Purity of IFN-a2 determined by SDS-polyacrylamide gel electrophoresis. The electrophoresis buffer was the standard Laemrnliz5system and the percentage of acrylamide 12%. The gel was stained with silver. The marker proteins (A) were lysozyme (14 kDa), soya bean trypsin inhibitor (21.5 kDa), carbonic anhydrase (31 kDa), ovalbumin (43 kDa), and serum albumin (68 kDa). The loading concentrations were (from left to rightl 100 (B), 5(I (C), 25 (D), 12.5 (E), 6.25 (F). 3.15 (G), 1.56 (H), 0.78 (11, and 0.39 (J)/zg per slot.
T h e yields and specific activities obtained at each step are s h o w n in the table. Properties of Purified H u - I F N - a 2 I n t e r f e r o n p r o d u c e d by this m e t h o d is o f high purity as j u d g e d by silver stained gel (Fig. 4) 25 and migrates as a single species with an apparent m o l e c u l a r weight o f 18,000 on S D S - p o l y a c r y l a m i d e gel e l e c t r o p h o r e sis. T h e h o m o g e n e i t y o f the p r e p a r a t i o n is suggested by the f o r m a t i o n o f crystals (Fig. 3). H o w e v e r , gel e l e c t r o p h o r e s i s by the m e t h o d o f Nav U. K. Laemmli, Nature (London) 227, 680 [1970).
174
PURIFICATION OF INTERFERONS
[24]
FIG. 5. Native PAGE of IFN-a2. The gel was prepared as described by Nakeshima and Makino. 26 The buffer used was the standard pH 4.0/3-alanine discontinuous system and the polyacrylamide gradient ranged from 5 to 30% (w/v). A is thyroglobulin (did not enter gel), B is IFN-c~2, and C is bovine growth hormone. The positions of several marker proteins run on the same gel are shown: 13 kDa is ribonuclease, 25 kDa is chymotrypsinogen, 43 kDa is ovalbumin, 68 kDa is bovine serum albumin, and 400 kDa is ferritin.
kashima and Makino 26 in the absence of ionic detergents shows smearing of the protein with apparent molecular weight distribution of 30,00060,000 (Fig. 5). This apparent absence of a species of homogeneous molecular radius under native conditions correlates with the anomalous elution behavior observed on gel filtration and with the reported sedimentation properties in the analytical ultracentrifuge, z7 The purified recombinant IFN-a2 behaved as a relatively hydrophobic molecule on C-18 reverse phase HPLC, eluting as a single major species 26 H. Nakashima and S. Makino, J. Biochem. (Tokyo) 88, 933 (19801. 27 S. Shire, Biochemistry 22, 2664 (1983).
[24]
PURIFICATIONOF HUMANIFN-a2
175
kd > I---...J Ld
O cO
p"l
Od
II
Ld
U
s°
Z 14 t . . . . . . .
cO Fr
"J
0
co rn
-k .....
o•~
ELUTION TIME
60min
FIG. 6. C-18 reverse p h a s e H P L C of IFN-a2. H P L C was performed with a Waters 6000A s y s t e m and a 4 × 250 m m S y n c h r o Pak C-18 analytical column ( S y n c h r o m Inc, Linden, Indiana). For this c h r o m a t o g r a m , 100/zg of protein was applied and the elution conditions were 0 to 35% acetonitrile in 20 rain, isocratic at 50% acetonitrile for 10 min, and finally a linear gradient to 75% acetonitrile in 10 rain. The flow rate was 1 ml/min and the mobile phase contained 0.1% (v/v) trifluoroacetic acid. The elution profile was monitored by absorbance at 280 nm, where 0.05 A_,s0unit represents m a x i m u m deflection.
at an acetonitrile concentration of approximately 55% (Fig. 6). (See also Staehelin et al. 28 and Felix et al. 29) Isoelectric focusing in agarose gels gave rise to multiple bands. The major component had a pl of 5.9 accompanied by three lesser anodic bands within 0.3 of a pl unit (Fig. 7). IFN-a2 prepared by this method had specific activity of 3.2 x 108 units/mg when assayed by CPE inhibition with HEp-2 cells and EMCV. Comments
The exploitation of gene cloning and in particular the development of high level expression systems in the bacterium E. coli has changed the nature of the problems involved in preparing homogeneous IFN-a. 2s T. Staehelin, D. S. Hobbs, H.-F. Kung, C.-Y. Lai, and S. Pestka, J. Biol. Chem. 256, 9750 (1981). -~ A. Felix, E. Heimer, and S. J. Tarnowski, this volume 133].
176
PURIFICATION OF INTERFERONS
[24]
®
@
Ld I-..3 LO t'~
LtJ U z
"..
-'-"
rn
rY
S rn <
/
1
O
I
2
I
I
I ,A]~
4
i pH O
~ 6
\
.O
\
I
8
I
I
10
ANODIC MIGRATION(CM) FIG. 7. isoelectric focusing of IFN-o~2. Thin layer agarose gels were poured with 3-10 pharmalytes (Pharmacia, Sweden) as a source of ampholytes. The focused gel was stained with Coomassie Blue, dried, and then scanned with a Joyce Loebel Chromoscan 3 densitometer.
Whereas isolation of IFN-c~ from naturally occurring sources involves a several thousand fold enrichment of starting material, recombinant techniques require only 3- to 10-fold purification to achieve homogeneity. The genetic engineering strategy must, however, solve a novel problem, namely the isolation, in the correct conformation, of an extracellular protein synthesized in an intracellular environment. The intracellular environment of microorganisms has a net reducing redox potential 3° and the formation of the two disulfide bridges of natural IFN-a2 is consequently not favored. Another complication which may be related to the synthesis of a reduced molecular species is the finding that during synthesis, the IFN-c~2 is laid down as insoluble aggregates in a morphologically distinct inclusion body. The challenge therefore is to solubilize this inclusion body and renature IFN-c~2 in the natural conformation with both disulfide bridges correctly formed. As we described above this aggregate was found to be soluble in strongly chaotropic solvents such as guanidine hydrochloride and could be renatured in high yield to an active conformation simply on dilution into aqueous solvents. The problem of formation of disulfide bridges from reduced interferon has been studied in detail elsewhere. 3~ 30 p. Apontoweil and W. Berends, Biochim. Biophys. Acta 339, I (1975). ~t H. Morehead. P. N. Johnston, and R. Wetzel, BiochemisoT 23, 2500 (19841.
[25]
RECOMBINANT
HUMAN
FIBROBLAST INTERFERON
177
This oxidation process occurs in the method described here either spontaneously or by copper catalyzed air oxidation during column chromatography on chelating sepharose. Also the fully oxidized form may be resolved from the other forms during purification. The separation of correctly refolded 1FN-a2 from contaminating conformational variants is not a trivial problem. These variants have similar properties to the native molecule and purification strategies are complicated by the tendency of 1FN-a2 to form oligomers transiently in concentrated solution (>1 mg/ml), z3 Conformational variants which participate in this association are consequently almost impossible to completely separate from the "native" conformer. The purification approach outlined above, incorporates separation techniques selected for their compatibility with buffer systems favoring the monomeric form of the protein: high ionic strength and low pH. The presence of conformational variants trapped in relatively concentrated preparations may contribute to the observed immunogenicily of the IFN-c~ preparations used in clinical trials. 32 Acknowledgments We wish to thank Dr. A. Pickett of the Fermentation Department and Dr. M. Hirschi of the Cell Biology Department, Biogen S.A. for their continued help on this project. We also wish to acknowledge the provision of the gel electrophoretic and HPLC data by Dr. Denis Bergher, Biogen S.A. ~'- P. W. Trown, M. J. Kramer, R. A. Dennin, E. V. Connell, A. V. Palleroni, J. Quesada, and J. U. Gutterman, Lam:et 81 (1983).
[25] P u r i f i c a t i o n o f R e c o m b i n a n t H u m a n F i b r o b l a s t I n t e r f e r o n P r o d u c e d in E s c h e r i c h i a coli By JOHN A. MOSCHERA, DIANA WOEHLE, KELLY PENG TSAI, C H I E N - H W A CHEN, a n d S. JOSEPH TARNOWSKI
Many interferons have been expressed in Escherichia coli. 1 Their use, however, requires purification to homogeneity so that traces of E. coli endotoxin and other contaminants are undetectable or at acceptable levS. Pestka, this volume [1].
METHODS IN ENZYMOLOGY, VOL. 119
Copyright © 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.
PURIFICATION OF INTERFERONS
[24]
[24] P u r i f i c a t i o n o f R e c o m b i n a n t H u m a n I F N - a 2 By DAVID R. THATCHER and NIKOS PANAYOTATOS
Alpha or leukocyte interferon represents a homologous family of proteins with antiviral activity secreted by nearly all virus-infected human cells. ~ Because in v i v o levels of synthesis are extremely low, interferon purification from natural sources has been a challenging technical problem. 2 Large volumes of either peripheral blood leukocyte sl-3 (buffy coats) or immortalized cell lines 4-~° are required in conjunction with sophisticated separation techniques such as reversed phase HPLC 2,J~ or monoclonal immunoaffinity chromatography. ~2,j3 The advent of gene technology has afforded a more efficient and cost effective approach to production of interferon. Microorganisms, expressing individual interferon genes at high level, 2 now provide an abundant source of protein for purification. As described in this chapter, purification of recombinant interferon can be achieved by standard methodology and can easily be scaled up to meet the precise and reproducible criteria required for manufacturing. In fact, human IFN-c~2 has been purified and crystallized on a commercial scale by Nagabushan and co-workers. J4 The Hu-IFN-a2 and the closely related Hu-IFN-c~A genes were isolated, respectively, by Nagata e t al. j5 and Streuli e t a l . j6 o n the one hand and by Maeda e t a/. ~7and Goeddel e t al. 18 on the other. The cDNA clones S. Pestka and S. Baron, this series, Vol. 78, p. 3. 2 S. Pestka, Arch. Biochem. Biophys. 221, I (1983). 3 K. Berg, C. A. Ogburn, K. Paucker, K. E. Mogensen, and K. Cantell, J. lmmunol. 114, 640 (1975). 4 H. Strander, K. E. Mogensen, and K. Cantell, J. C/in. Mierobiol. 1, 116 (1975). 5 A. Mizrahi, this series, Vol. 78, p. 54. 6 G. Bodo, this series, Vol. 78, p. 69. 7 F. Klein and R. T. Ricketts, this series, Vol. 78, p. 75. 8 p. C. Familletti, L. Costello, C. A. Rose, and S. Pestka, this series, Vol. 78, p. 83. 9 T. M. Powledge, Bio/Technology 214 (1984). to A. W. Phillips, N. B. Finter, C. J. Burman, and G. D. Ball, this volume [4]. H M. Rubinstein, S. Rubinstein, P. C. Familletti, R. S. Miller, A. A. Waldman, and S. Pestka, Proe. Natl. Aead. Sci. U.S.A. 76, 640 (1979). iz D. Secher and D. C. Burke, Nature (London) 285, 446 (1980). ~3 T. Staehelin, D. S. Hobbs, H.-F. Kung, and S. Pestka, this series, Vol. 78, p. 505. 14 T. L. Nagabushan and P. Leibowitz, in "Interferon alpha 2: preclinical and clinical evaluation," pp. 1-12. Nijhoff, The Hague, 1985. t5 S. Nagata, H. Taira, A. Hall, L. J o h n s r u d , M. Streuli, J. Escodi, W. Boll, K. Cantell, and C. W e i s s m a n n , Nature (London) 284, 316 (1980).
M E T H O D S IN E N Z Y M O L O G Y , VOL. 119
Copyright © 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.
[24]
PURIFICATION OF HUMANIFN-a2
~x~o2O's~
V
167
Cene
pNKS97J
Ori ~> FIO. 1. Structure of the pl.TT.a2.copl expression vector, pI.TT.c~2.cop! consists of the pBR322 sequence between the unique SalI (position 650) and EcoRl (position 4359) restriction sites and 112 bp of bacteriophage T7 DNA containing the ribosome binding site for gene 1.1.19 The promoter pl described in this work introduced at the EcoRI site and the human leukocyte IFN-a2 gene at the Sail site. The positions of the unique PstI and AvaI restriction sites are shown. The origin and direction of replication, the positions of the primer RNA (Pm) and the RNA I (pl) promoters, as well as the copl point mutation at position 3029 of the pBR322 map are also indicated.
were prepared from mRNA derived from human leukocytes induced with Sendai virus. The region coding for the natural interferon signal peptide (which cannot be processed correctly by Escherichia coli) was removed, and the gene sequence corresponding to the extracellular protein was expressed in E. coli. ~8 The Plasmid Vector
A plasmid containing the IFN-a2 gene was kindly provided by C. Weissmann. A 720 bp H i n d I I I - P s t I fragment was digested with S 1 nuclease to provide blunt ends and was inserted into plasmid pNKS97 at a unique SalI site rendered blunt with SI nuclease (Fig. 1). Plasmid
ii, M. Slreuli, S. Nagata, and C. Weissmann, Science 209, 1343 (1980). ~7 S. Maeda, R. McCandliss, M. Gross, A. Sloma, P. C. Familletli, J. M. Tabor, M. Evinger, W. P. Levy, and S. Pestka, Proc. Natl. Acad. Sci. U.S.A. 77, 7010 (1980); 78, 4648 (1981). 's D. V. Goeddel, E. Yelverton, A. UIIrich, H. L. Heyneker, G. Miozzari, W. Holmes, P. H. Seeburg, T. Dull, L. May, N. Slebbing, R. Crea, S. Maeda, R. McCandliss, A. Sloma, J. M. Tabor, M. Gross, P. C. Familletti, and S. Peslka, Nature (London) 287, 411 (1980).
168
PURIFICATION OF INTERFERONS
[24]
pNKS97 is a versatile vector for gene expression in E. coli. 19This plasmid consists of 3709 bp of the pBR322 sequence between the SalI and EcoRI sites containing the/3-1actamase (amp R) and replication regions as well as a 112 bp fragment from bacteriophage T7 which contains the ribosome binding site for gene 1.1 (Fig. 1). A unique EcoRI site allows the insertion of promoter fragments upstream from the ribosome binding site whereas the unique SalI site permits the insertion of the gene to be expressed (in this case the IFN-a2 gene) in phase with an initiating methionine codon. The promoter used was the PI promoter from ColE1 which promotes the transcription of a small RNA molecule (RNAI) which is involved in the control of plasmid replication. A copy number mutation of this plasmid [pI.T7.a2.copl] was isolated by selection on L-broth agar plates containing 20 mg/ml methicillin. 2° Fermentation The bacteria harboring the pI.T7.a2.copl plasmid were maintained on L-agar plates containing l0 mg/ml ampicillin and were subcultured at weekly intervals. Cultures were allowed to grow at 37 ° for 20 hr into stationary phase in overfilled 31 shake flasks containing 1.6 liter of standard L-broth at low agitation. IFN-a2 expression was of the order of 515 × 109 units/liter/OD590 (16-47 mg/liter), or 10-30% of total cellular protein. Purification of IFN-a2 Increased levels of interferon expression in this system lead to a proportionately increasing fraction of interferon protein laid down as an intracellular insoluble aggregate (Fig. 2). At high levels of expression, over 95% of the interferon synthesized by the pI.T7.a2.copl system is in an insoluble physical state. This aggregation of foreign or mutant proteins expressed at high levels in E. coli is a well documented phenomenon 21,22 and can be exploited to advantage in protein purification strategies. Upon cell breakage, the soluble cell constituents, which include the majority of the contaminating E. coli proteins, can be washed away by successive 19 N. Panayotatos and K. Truong, Nucleic Acids Res. 9, 5679 (1981). 20 N. Panayotatos, A. Fontaine, and K. Truong, J. Cell. Biochem., Suppl. 7B, Abstr. No. 765 (1983). 2J A. L. Goldberg and A. C. St. John, Annu. Rev. Biochem. 45, 747 (1976). 22 D. C. Williams, R. M. van Frank, W. L. Muth, and J. P. Burnen, Science 215,687 (1982).
[24]
PURIFICATIONOF HUMAN IFN-o~2 10.C
169
o
i
z LL W
_J
0 0
C) .J 0 V)
0 0
W (.9 1.0
0
0 0
0
0
Z ul U W Q_ 00 0
(.9 0 ._J
0.1C 7.0
I
I
8.0
90
L O G SPECIFIC A C T I V I T Y
0
o
I
10.0 {U/GRAM)
FIG. 2. The fraction of soluble intracellular 1FN-c~2 at different levels of e x p r e s s i o n . Total interferon expression (abscissa) was determined by bioassay of cells lysed in 0 . 1 % S D S a n d is expressed in terms of units per gram fresh weight o f E. coli cells. To determine
the fraction of soluble interferon, cells were first sheared with a french press and then centrifuged at 10000 g for 1 hr. The supernatant was then bioassayed and the resulting value expressed as a p e r c e n t a g e o f the total interferon produced.
resuspension and centrifugation steps. Subsequently, the insoluble contaminating membrane proteins of this washed pellet may be selectively solubilized to leave an insoluble pellet whose major proteinaceous constituent is IFN-o~2. The interferon aggregate is then solubilized by a chaotropic solvent and renatured to give a near homogeneous solution of IFN-a2 which can be purified by ion-exchange, metal chelate, and gel permeation chromatography, followed by recrystallization.
Buffers Buffer A: 0. I M Tris. HCI, 0.05 M EDTA, pH 7.5 Buffer B: 2 M guanidine. HC1 treated with activated charcoal and filtered through a 0.22-/~m filter Buffer C: 8 M guanidine. HC1, purified as above Buffer D: 0.04 M sodium dihydrogen phosphate Buffers E and F as D but adjusted to pH 5.5 and 6.0, respectively, with 1 M sodium hydroxide.
170
PURIFICATION OF INTERFERONS
[24]
Cell Breakage The inherent advantage offered by the observed partitioning of IFNa2 into inclusion bodies can only be exploited fully if near total cell breakage is achieved. As the cells are fermented into late stationary phase and are consequently difficult to rupture by mechanical shear, pretreatment with lysozyme has been adopted in order to destroy the integrity of the peptidoglycan layer of the cell wail. This treatment, as well as increasing the efficiency of cell breakage, also reduces the level of carbohydrate in the final washed pellet. The method described below is designed for 100 g to 1 kg of cell pellet. Cells were harvested from fermentation broth by centrifugation at 5000 g. The cells were weighed and placed in a chilled Waring Blendor. A 2-fold (w/v) excess of Buffer A was added, with blending at low speed for 30 sec. Solid sucrose was then dissolved in the cell suspension to give a final concentration of 30% (w/v) followed by the addition of 1 mg of lysozyme hydrochloride for each gram of original cell pellet. After incubation for 1 hr at 30°, the cell suspension was diluted with an equal volume of chilled deionized water. The viscous suspension was thinned by blending with a Kinematica Polytron PT45 and passed once through a french press at a pressure of 800 bar (about 12,000 Ib/infl). The solid components of the suspension, interferon aggregate and cell membrane, were then collected by centrifugation at 10000 g for 30 min. The pellet was resuspended in Buffer B and recentrifuged. This washing procedure was then repeated two more times.
Extraction The final washed pellet was solubilized in a 10-fold (w/v) excess of Buffer C and immediately diluted 4-fold with Buffer A. The suspension was then centrifuged at 10000 g for 30 min and the supernatant dialyzed four times against 20 volumes of distilled water at 4° over a 24-hr period. The cloudy brown precipitate that formed was sedimented at 10000 g for 30 min, the precipitate discarded, and the clear colorless supernatant collected.
Column Chromatography The IFN-o~2 at this stage comprised >90% of the protein in solution as judged by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on samples reduced with 2-mercaptoethanol. Nonreduced samples, however, run on the same SDS-PAGE system, showed a doublet band; the
[24]
PURIFICATIONOF HUMANIFN-a2
171
upper band comigrated with reduced interferon and the lower band with native oxidized interferon. 23 The pH of the solution was reduced to 4.0 by the addition of concentrated orthophosphoric acid and the solution applied to a 5 x 10 cm column of SP Sephadex A-50 equilibrated with Buffer D. The column was then washed with 1 liter of Buffer D followed by 500 ml each of Buffers E and F. Fractions (50 ml) were collected and those containing interferon, as judged by SDS-PAGE, were pooled and passed through a sintered glass funnel containing a bed of DEAE-Sephadex A20 (5 × 10 cm diameter) equilibrated with Buffer F. The flow through from this column was then adjusted to 1 M with respect to NaCI concentration and applied to a copper chelate column. A column (2.5 x 5 cm) of chelating Sepharose 6B (Pharmacia, Uppsala) was prepared as described by the manufacturer. After washing with 9 bed volumes of 1 M NaCI the column was charged with C u 2+ ions 24 by applying a solution of I mg/mi copper sulfate. The column was then repacked with I cm of unchelated matrix at the bottom of the column to mop up any leached copper ion. The column was then washed successively with Buffers F, E, and D all containing 1 M NaCI. Fractions (50 ml) were collected and those containing oxidized interferon as judged by SDS-PAGE run under nonreducing conditions were pooled. The pooled solution was raised to 80% ammonium sulfate saturation by the addition of the solid. The flocculent white precipitate which formed was collected by centrifugation and redissolved in a minimum volume of Buffer F. The concentrate was then applied to a 5 x 100 cm column of Sephacryl S-200 equilibrated with Buffer D containing 1 M NaCI. Fractions (5 ml) were collected and the elution profile monitored at 280 nm. The protein peak eluted as an asymmetric peak with a retention volume corresponding to a molecular weight of approximately 40,000 to 60,000. The major portion of this peak was pooled and the ammonium sulfate saturation raised to 80%. The white precipitate which formed was collected by centrifugation and suspended in a minimum volume of Buffer F. The suspension was then dialyzed against 5 liters of buffer F diluted 100fold. The precipitate disappeared within 30 min and after 48 hr the dialysis bag was filled with microcrystals (Fig. 3). These crystals were collected by low speed centrifugation in a bench centrifuge and stored moist at 4°.
~.3 S. Pestka, B. Kelder, D. K. Tarnowski, and S. J. Tarnowski, Anal. Biochem. 132, 328 (1983). 24 K. C. Chada, this series, Vol. 78. p. 220.
FIG. 3. P h a s e - c o n t r a s t m i c r o s c o p y of I F N - a 2 m i c r o c r y s t a l s . Magnification, x 100. PURIFICATION OF IFN-c~2"
Stage Suspended cells Cell homogenate Supernatant Supernatant of T w e e n w a s h Dialysed guanidine extract Supernatant SP-Sephadex eluate Combined Cu-chelate eluate Combined Sephacryl 5200 fractions Crystallized
Yield (%)
Specific activity (units/mg)
1.6 x 1012
100
2.9 x 106
1.1 × 109
3.7 x 1012
--
9.6 × 107
3140 1080
1.2 x 10s 1.1 x 107
3.7 x 10 H 1.2 × 10 I°
23 0.8
1.5 x 107 5.9 x 106
3160
1.2 x 108
3.7 x 1011
23
--
3060 550
1.2 x 10s 1.1 × 109
3.7 x 10 H 6 x I0 II
23 38
---
500
9.3 x 108
1.4 x 1012
87
--
308
8 x 108
2.4 x 1011
15
--
612
2.7 x lO8
1.7 x lO 11
11
3.2 x 10s
Volume (ml)
Activity/ml (units/ml)
Total (units)
1500
1.1 x l09
3400
A b o u t 770 g of E. coli containing I F N - a 2 was used as the starting material.
[24]
PURIFICATION OF HUMAN 1FN-a2
173
FIG. 4. Purity of IFN-a2 determined by SDS-polyacrylamide gel electrophoresis. The electrophoresis buffer was the standard Laemrnliz5system and the percentage of acrylamide 12%. The gel was stained with silver. The marker proteins (A) were lysozyme (14 kDa), soya bean trypsin inhibitor (21.5 kDa), carbonic anhydrase (31 kDa), ovalbumin (43 kDa), and serum albumin (68 kDa). The loading concentrations were (from left to rightl 100 (B), 5(I (C), 25 (D), 12.5 (E), 6.25 (F). 3.15 (G), 1.56 (H), 0.78 (11, and 0.39 (J)/zg per slot.
T h e yields and specific activities obtained at each step are s h o w n in the table. Properties of Purified H u - I F N - a 2 I n t e r f e r o n p r o d u c e d by this m e t h o d is o f high purity as j u d g e d by silver stained gel (Fig. 4) 25 and migrates as a single species with an apparent m o l e c u l a r weight o f 18,000 on S D S - p o l y a c r y l a m i d e gel e l e c t r o p h o r e sis. T h e h o m o g e n e i t y o f the p r e p a r a t i o n is suggested by the f o r m a t i o n o f crystals (Fig. 3). H o w e v e r , gel e l e c t r o p h o r e s i s by the m e t h o d o f Nav U. K. Laemmli, Nature (London) 227, 680 [1970).
174
PURIFICATION OF INTERFERONS
[24]
FIG. 5. Native PAGE of IFN-a2. The gel was prepared as described by Nakeshima and Makino. 26 The buffer used was the standard pH 4.0/3-alanine discontinuous system and the polyacrylamide gradient ranged from 5 to 30% (w/v). A is thyroglobulin (did not enter gel), B is IFN-c~2, and C is bovine growth hormone. The positions of several marker proteins run on the same gel are shown: 13 kDa is ribonuclease, 25 kDa is chymotrypsinogen, 43 kDa is ovalbumin, 68 kDa is bovine serum albumin, and 400 kDa is ferritin.
kashima and Makino 26 in the absence of ionic detergents shows smearing of the protein with apparent molecular weight distribution of 30,00060,000 (Fig. 5). This apparent absence of a species of homogeneous molecular radius under native conditions correlates with the anomalous elution behavior observed on gel filtration and with the reported sedimentation properties in the analytical ultracentrifuge, z7 The purified recombinant IFN-a2 behaved as a relatively hydrophobic molecule on C-18 reverse phase HPLC, eluting as a single major species 26 H. Nakashima and S. Makino, J. Biochem. (Tokyo) 88, 933 (19801. 27 S. Shire, Biochemistry 22, 2664 (1983).
[24]
PURIFICATIONOF HUMANIFN-a2
175
kd > I---...J Ld
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II
Ld
U
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-k .....
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ELUTION TIME
60min
FIG. 6. C-18 reverse p h a s e H P L C of IFN-a2. H P L C was performed with a Waters 6000A s y s t e m and a 4 × 250 m m S y n c h r o Pak C-18 analytical column ( S y n c h r o m Inc, Linden, Indiana). For this c h r o m a t o g r a m , 100/zg of protein was applied and the elution conditions were 0 to 35% acetonitrile in 20 rain, isocratic at 50% acetonitrile for 10 min, and finally a linear gradient to 75% acetonitrile in 10 rain. The flow rate was 1 ml/min and the mobile phase contained 0.1% (v/v) trifluoroacetic acid. The elution profile was monitored by absorbance at 280 nm, where 0.05 A_,s0unit represents m a x i m u m deflection.
at an acetonitrile concentration of approximately 55% (Fig. 6). (See also Staehelin et al. 28 and Felix et al. 29) Isoelectric focusing in agarose gels gave rise to multiple bands. The major component had a pl of 5.9 accompanied by three lesser anodic bands within 0.3 of a pl unit (Fig. 7). IFN-a2 prepared by this method had specific activity of 3.2 x 108 units/mg when assayed by CPE inhibition with HEp-2 cells and EMCV. Comments
The exploitation of gene cloning and in particular the development of high level expression systems in the bacterium E. coli has changed the nature of the problems involved in preparing homogeneous IFN-a. 2s T. Staehelin, D. S. Hobbs, H.-F. Kung, C.-Y. Lai, and S. Pestka, J. Biol. Chem. 256, 9750 (1981). -~ A. Felix, E. Heimer, and S. J. Tarnowski, this volume 133].
176
PURIFICATION OF INTERFERONS
[24]
®
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ANODIC MIGRATION(CM) FIG. 7. isoelectric focusing of IFN-o~2. Thin layer agarose gels were poured with 3-10 pharmalytes (Pharmacia, Sweden) as a source of ampholytes. The focused gel was stained with Coomassie Blue, dried, and then scanned with a Joyce Loebel Chromoscan 3 densitometer.
Whereas isolation of IFN-c~ from naturally occurring sources involves a several thousand fold enrichment of starting material, recombinant techniques require only 3- to 10-fold purification to achieve homogeneity. The genetic engineering strategy must, however, solve a novel problem, namely the isolation, in the correct conformation, of an extracellular protein synthesized in an intracellular environment. The intracellular environment of microorganisms has a net reducing redox potential 3° and the formation of the two disulfide bridges of natural IFN-a2 is consequently not favored. Another complication which may be related to the synthesis of a reduced molecular species is the finding that during synthesis, the IFN-c~2 is laid down as insoluble aggregates in a morphologically distinct inclusion body. The challenge therefore is to solubilize this inclusion body and renature IFN-c~2 in the natural conformation with both disulfide bridges correctly formed. As we described above this aggregate was found to be soluble in strongly chaotropic solvents such as guanidine hydrochloride and could be renatured in high yield to an active conformation simply on dilution into aqueous solvents. The problem of formation of disulfide bridges from reduced interferon has been studied in detail elsewhere. 3~ 30 p. Apontoweil and W. Berends, Biochim. Biophys. Acta 339, I (1975). ~t H. Morehead. P. N. Johnston, and R. Wetzel, BiochemisoT 23, 2500 (19841.
[25]
RECOMBINANT
HUMAN
FIBROBLAST INTERFERON
177
This oxidation process occurs in the method described here either spontaneously or by copper catalyzed air oxidation during column chromatography on chelating sepharose. Also the fully oxidized form may be resolved from the other forms during purification. The separation of correctly refolded 1FN-a2 from contaminating conformational variants is not a trivial problem. These variants have similar properties to the native molecule and purification strategies are complicated by the tendency of 1FN-a2 to form oligomers transiently in concentrated solution (>1 mg/ml), z3 Conformational variants which participate in this association are consequently almost impossible to completely separate from the "native" conformer. The purification approach outlined above, incorporates separation techniques selected for their compatibility with buffer systems favoring the monomeric form of the protein: high ionic strength and low pH. The presence of conformational variants trapped in relatively concentrated preparations may contribute to the observed immunogenicily of the IFN-c~ preparations used in clinical trials. 32 Acknowledgments We wish to thank Dr. A. Pickett of the Fermentation Department and Dr. M. Hirschi of the Cell Biology Department, Biogen S.A. for their continued help on this project. We also wish to acknowledge the provision of the gel electrophoretic and HPLC data by Dr. Denis Bergher, Biogen S.A. ~'- P. W. Trown, M. J. Kramer, R. A. Dennin, E. V. Connell, A. V. Palleroni, J. Quesada, and J. U. Gutterman, Lam:et 81 (1983).
[25] P u r i f i c a t i o n o f R e c o m b i n a n t H u m a n F i b r o b l a s t I n t e r f e r o n P r o d u c e d in E s c h e r i c h i a coli By JOHN A. MOSCHERA, DIANA WOEHLE, KELLY PENG TSAI, C H I E N - H W A CHEN, a n d S. JOSEPH TARNOWSKI
Many interferons have been expressed in Escherichia coli. 1 Their use, however, requires purification to homogeneity so that traces of E. coli endotoxin and other contaminants are undetectable or at acceptable levS. Pestka, this volume [1].
METHODS IN ENZYMOLOGY, VOL. 119
Copyright © 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.