- Email: [email protected]
[28] Nickel chelate chromatography of human immune interferon
[28]
HUMAN IMMUNEINTERFERON
199
Acknowledgments I wish to thank Ms. Pat Gregory for her skillful secretarial assistance. ! would also like to thank Drs. W. D. Terry and C. G. Smith and Mr. D. Lewis for their support and encouragement.
~4y . H. Tan, F. Barakat, W. Berthold, H. Smith-Johannsen, and C. Tan, J. Biol. Chem. 254, 8067 (1979). 15 H.-J. Friesen, S. Stein, M. Evinger, P. C. Familletti, J. Moschera, J. Meienhofer, J. Shively, and S. Pestka, Arch. Biochem. Biophys. 206, 432 (1981).
[28] N i c k e l C h e l a t e C h r o m a t o g r a p h y o f Human Immune Interferon
By DORIAN H. COPPENHAVER Introduction
Since its introduction by Porath and colleagues 1,2 metal chelate chromatography has been successfully applied to the purification of a variety of proteins. The technique was quickly applied to interferon, with human fibroblast interferon being successfully chromatographed on Zn 2+ chelate columns. 3 Neither human I F N - y nor I F N - a is adsorbed to Zn 2+ chelates under standard conditions. 3 Very different results are obtained when a Cu z+ charged metal chelate column is used for chromatography of interferons. Cu 2+ chelates are more retentive than are Zn 2+ chelates2; practically all serum proteins are strongly adsorbed to Cu 2+ chelate columns. 4 H u m a n a a n d / 3 interferons are all strongly adsorbed to Cu 2+ chelates, which are eluted from the columns only with difficulty. 5 Human IFN-y is also strongly adsorbed to Cu z+ chelates, 6 but does not interact with most 1 L. Lundberg and J. Porfith, J. Chromatogr. 90, 87 (1975). 2 j. Porfith, J. Carlsson, I. Olsson, and G. Belfrage, Nature (London) 258, 598 (1975). 3 V. G. Edy, A. Billiau, and P. DeSomer, J. Biol. Chem. 252, 5934 (1977). 4 D. H. Coppenhaver, N. P. Sollenne, and B. H. Bowman, Arch. Biochem. Biophys. 226, 218 (1983). 5 K. C. Chada, P. M. Grob, A. J. Mikulski, L. R. Davis, Jr., and E. Sulkowski, J. Gen. Virol. 43, 701 (1979). 6 E. Sulkowski, K. Vastola, D. Oleszek, and W. von Meunchhausen, in "Affinity Chromatography and Related Techniques" (T. C. J. Gribnau, J. Visser, and R. J. F. Nivard, eds.), p. 313. Elsevier, Amsterdam, 1982.
METHODS IN ENZYMOLOGY.VOL. 119
Copyright© 1986by AcademicPress, Inc. All rightsof reproductionin any formreserved.
200
PURIFICATION OF INTERFERONS
[28]
other metal chelates, which Porath 2 has determined to be weaker adsorbents. 7 Human IFN-y is retained on iminodiacetic acid activated agarose columns which have been charged with Ni 2+, however. This moderately weak metal chelate gel shows an unexpected interaction with IFN-y, the mechanism of which has not been determined, which can be useful in its purification. Materials Sepharose 6B, Pharmacia Fine Chemicals 1,4-Butanediol diglycidylether, Aldrich Chem. Co. Iminodiacetic acid disodium salt, Aldrich Chem. Co. Sodium Borohydride, Sigma NiSO4" 7H20 (Gold Label), Aldrich Chem. Co. Tris(hydroxymethyl) aminomethane NazCO3 EDTA NaCI NaC2H302 NaHzPO4 Solutions 0.02 M Tris. HCI, 0.15 M NaC1, pH 8.5 (Buffer A) 0.1 M NaH2PO4, 0.15 M NaCI, pH 6.5 (Buffer B) 0.1 M NaCzH302, 1.0 M NaCI, pH 4.5 (Buffer C) 0.05 M EDTA, 1.0 M NaCI Interferon and Interferon Assay Human immune interferon was prepared from peripheral blood lymphocytes which had been stimulated with staphylococcal enterotoxin A (SEA) as previously described, s,9 Interferon was concentrated by chromatography on columns of silicic acid and eluted with 0.3 M (CH3)4NCI in PBS. J0 In some experiments, silicic acid concentrated interferon was further purified by gel filtration on Ultragel AcA54 eluted with 1.0 M NaC1 in PBS before Ni 2÷ chelate chromatography was performed. 1° Interferon 7D. H. Coppenhaver, unpublished observations. s M. P. Langford,J. A. Georgiades,G. J. Stanton, F. Dianzani, and H. M. Johnson, Infect. Immun. 26, 36 (1979). 9 H. M. Johnson, F. Dianzani, and J. A. Georgiades, this series, Vol. 78, p. 158. ~0V. M. Papermasterand S. Baron, Tex. Rep. Biol. Med. 41, 672 (1981-1982).
[28]
HUMAN IMMUNE INTERFERON
201
was assayed on human WISH cells by estimation of reduction of cytopathic effectU; interferon activity was expressed in terms of National Institutes of Health reference standard. P r e p a r a t i o n of I m i n o d i a c e t i c A c i d - A c t i v a t e d S e p h a r o s e an d Ni 2+ Chelate Column
Sepharose 6B is epoxy activated essentially as described by Porath.l,2 Swelled resin (150 ml) is dried in air (30 min on a sintered glass filter) before being added to a mixture of 100 ml 1,4-butanediol diglycidyl ether and 200 mg of NaBH4 in 100 ml of 0.5 M NaOH. The mixture is sealed and incubated with gentle agitation in a 25° water bath for 24 hr. The resin is then removed and exhaustively washed with 100-200 volumes of deionized water. The washed resin is suction dried on a sintered glass filter before being added to 20 g of iminodiacetic acid, disodium salt, dissolved in 100 ml of 2 M Na2CO3. The resin/iminodiacetic acid mixture is then placed in a 65 ° water bath and incubated with constant gentle agitation for 24 hr. After incubation, the iminodiacetic acid activated Sepharose 6B is exhaustively washed (100-200 volumes) with deionized water after which it is ready for chromatographic use. Columns of epoxy activated Sepharose 6B which have not been previously used are flushed with 0.05 M EDTA in 1.0 M NaCI to remove any metal ions which have been inadvertently bound to the resin. The EDTA is then flushed from the column with Buffer A before charging the resin w i t h N i 2+. The column is charged by passing a 2-3 mg/ml solution of NiSO4 in water or Buffer A through the column until the top 80% of the resin is completely saturated. The resin which has complexed with Ni 2+ will be pale blue; the uncomplexed resin serves as a reservoir to scavenge Ni 2+ ions which are displaced during the course of the chromatography. Finally, the column is reequilibrated with Buffer A before sample application. Nickel Chelate Chromatography Samples of partially purified immune interferon that have been dialyzed against Buffer A are loaded onto the column which is then developed at a linear flow rate of 10-15 ml/cm2/hr at 22°. The volume of the applied sample does not appear to be critical. The column is flushed with 4-5 column volumes of Buffer A, until unbound material is totally eluted. An intermediate pH buffer (Buffer B) may then be applied to the column u p. C. Familletti, S. Rubinstein, and S. Pestka, this series, Vol. 78, p. 387.
202
PURIFICATION OF INTERFERONS
[28]
QUANTITATIVE DATA ON NICKEL CHELATE CHROMATOGRAPHY OF HUMAN I F N - ' y
Sample Expt. A Silicic acid concentrated IFN-3, Eluate from Ni2+ column Peak fraction Expt. B AcA-54 purified IFN-'y Eluate from Ni-'+ column Peak fraction Expt. C AcA-54 purified IFN-~/ Eluate from Ni2+ column Peak fraction
Total units
Specific activity
Yield(%)
Purification factor
2.0 x 103
8.0 × 103
1.0 x 103
--
--
8.7
0.9 x l0~
7.8 × 103
7.8 x 104
98
78
2.9
2.0 x 103
5.8 x 103
2.0 x l0 s
72
200
9.2
3.0 x 103
2.7 × 104
4.3 x 103
--
--
2.5
X 10 3
3.0 X l04
1.7 X 10"
108
395
4.0
5.0 x 103
2.0 x 104
5.0 x 106
74
1,163
29.0
3.0 × 103
2.7 x 104
4.0 x 104
--
14.4
3.2 x 103
4.6 x 104
1.6 x l06
53
40
2.4
8.0 x l0 t
1.9 x 104
8.9 x 106
41
222
Volume (ml)
Units/ml
4.0
12.0
--
to r e m o v e w e a k l y a d s o r b e d m a t e r i a l . I n t e r f e r o n a c t i v i t y is r e c o v e r e d b y flushing with 4 - 5 c o l u m n v o l u m e s o f Buffer C. T h e e n t i r e c h r o m a t o g r a p h i c p r o c e d u r e m a y b e c a r r i e d o u t at 4°; h o w e v e r , slightly b e t t e r resol u t i o n a n d r e c o v e r y o f i n t e r f e r o n a c t i v i t y are o b t a i n e d w h e n the c o l u m n is m a i n t a i n e d at r o o m t e m p e r a t u r e . T h e c o l u m n effluent s h o u l d be m a i n t a i n e d at 4 ° to p r e s e r v e i n t e r f e r o n activity. T h e IFN-3, does n o t s e e m to be a d v e r s e l y affected b y s h o r t (24 hr) e x p o s u r e to the p H 4.5 buffer at 4 °. T o t a l r e c o v e r y o f a p p l i e d i n t e r f e r o n a c t i v i t y has r a n g e d f r o m 53 to 125% in v a r i o u s e x p e r i m e n t s ; g r e a t e r t h a n 90% r e c o v e r y is typical. F r o m 80 to 96% o f the r e c o v e r e d I F N - y a c t i v i t y is a s s o c i a t e d with the m a t e r i a l eluting at p H 4.5. T h e r e m a i n d e r is f o u n d in the initial w a s h . T y p i c a l r e s u l t s f r o m N i 2÷ c h e l a t e c h r o m a t o g r a p h y o f I F N - y p r e p a r a t i o n s are g i v e n in the table, a n d a r e p r e s e n t a t i v e c o l u m n profile is d e p i c t e d in Fig. 1.
Comments C h r o m a t o g r a p h y o n c o l u m n s o f e p o x y a c t i v a t e d a g a r o s e c h a r g e d with Ni 2÷ ions has p r o v e n to be a useful step in I F N - y purification. T h e tech-
[28]
HUMAN IMMUNE INTERFERON ,
~
,
203
10,000
o.15
E
tO
z 0.10
5000
to~
0 e.~
3
<
0.05
200 o.01 o
10
20
30
Fraction No.
FIG. 1. Nickel chelate chromatography of silicic acid concentrated human IFN-% A volume of 4.0 ml of concentrated, dialyzed IFN-3, was applied to a 1.5 × 12 cm column of iminodiacetic acid-activated Sepharose 6B which had been charged with Ni -'+ and equilibrated in 20 mM Tris. HCI, 0.15 M NaC1, pH 8.5. After 20 fractions of 2.9 ml had been collected, the elution buffer was changed to 0.1 M NaC2H302, 1.0 M NaCI, pH 4.5. Units of IFN activity (dashed line) and total UV absorbance (solid line) are shown. Quantitative data for this experiment are given in the table (Expt. A).
nique in general appears to be forgiving, in that useful results have been obtained under a variety of conditions. Experience has shown that some precautions will enhance the success of the technique. Although chromatography can be carried out under cold room conditions, total recovery of IFN-y activity is enhanced if the column is maintained at 22°. No deterioration of the separation resin has been observed after dozens of experiments, but results are enhanced if the column is stripped of bound metal ions with 50 mM EDTA and recharged with Ni 2÷ after each run. As might be expected theoretically, ~,2 maintenance of a basic pH (8.0-8.5) appears to be important to maximize the initial adsorption of the IFN-y to the Ni 2+ chelate column. Adsorption to metal chelates is effective at a wide range of ionic strengths, but appears to undergo a minimum at about 1 M NaCI. 12 Hence, an increased appearance of IFN activity in the wash fraction can be expected if the sample is loaded in buffers containing 1 M J2 j. Porftth and B. Olin,
Biochemistry 22, 1621 (1983).
204
PURIFICATION
OF INTERFERONS
[29]
NaCI or maintained at neutral pH. Sample volume, however, does not appear to be a critical variable. Although slightly better results have been obtained with more concentrated samples, even dilute preparations have been successfully chromatographed on these columns. In these cases (e.g., Expt. C, the table) a significant concentration of the IFN-y activity has been obtained, particularly in peak fractions. Thus, Ni 2+ chelate chromatography may be a useful technique for both purification and concentration of IFN-y.
[29] 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
Human Immune Interferon
B y H S I A N G - F U K U N G , Y U - C H I N G E . PAN, JOHN MOSCHERA, KELLY TSAI, EVA BEKESI, MAY CHANG, HIROMU SUGINO, a n d SusuMu HONDA
Full length cDNA coding for mature human immune interferon (IFNy) has been isolated and the coding sequence for mature IFN-y has been expressed in Escherichia coli, j-3 yeast, 4 and monkey cells. 1,2 We have purified recombinant immune interferon (IFN-y) produced in E. coli. The purification as well as structural characterization of the bacterial product is described here. Materials and General Procedures Bacteria and Bacterial Fermentation Escherichia coli RR1 (pRK248clts, pRC231/IFN-7) was used for fermentations to produce IFN-y. 3 pRC231/IFN-7 differs from pRC23/IFN3/3 in nucleotide spacing between the Shine-Dalgaro sequence and the ATG initiation codon, pRC2314a is a derivative of pBR322 containing the phage h PL promoter. Expression of the IFN-'y gene is controlled by the P. W. Gray, D. W. Leung, D. Pennica, E. Yelverton, R. Najarian, C. C. Simonsen, R. Derynck, P. J. Sherwood, D. M. Wallace, S. L. Berger, A. D. Levinson, and D. V. Goeddel, Nature (London)295, 503 (1982). 2 R. Devos, H. Cheroutre, Y. Taya, W. Degrave, H. van Heuverswyn, and W. Fiers, Nucleic Acids Res. 10, 2487 (1982). 3 R. Crowl, this volume [55]. 4 R. Derynck, A. Singh, and D. V. Goeddel, Nucleic Acids Res. U , 1819 (1983). 4~ R. Crowl et al. In preparation.
METHODS IN ENZYMOLOGY, VOL. 119
Copyright © 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.
HUMAN IMMUNEINTERFERON
199
Acknowledgments I wish to thank Ms. Pat Gregory for her skillful secretarial assistance. ! would also like to thank Drs. W. D. Terry and C. G. Smith and Mr. D. Lewis for their support and encouragement.
~4y . H. Tan, F. Barakat, W. Berthold, H. Smith-Johannsen, and C. Tan, J. Biol. Chem. 254, 8067 (1979). 15 H.-J. Friesen, S. Stein, M. Evinger, P. C. Familletti, J. Moschera, J. Meienhofer, J. Shively, and S. Pestka, Arch. Biochem. Biophys. 206, 432 (1981).
[28] N i c k e l C h e l a t e C h r o m a t o g r a p h y o f Human Immune Interferon
By DORIAN H. COPPENHAVER Introduction
Since its introduction by Porath and colleagues 1,2 metal chelate chromatography has been successfully applied to the purification of a variety of proteins. The technique was quickly applied to interferon, with human fibroblast interferon being successfully chromatographed on Zn 2+ chelate columns. 3 Neither human I F N - y nor I F N - a is adsorbed to Zn 2+ chelates under standard conditions. 3 Very different results are obtained when a Cu z+ charged metal chelate column is used for chromatography of interferons. Cu 2+ chelates are more retentive than are Zn 2+ chelates2; practically all serum proteins are strongly adsorbed to Cu 2+ chelate columns. 4 H u m a n a a n d / 3 interferons are all strongly adsorbed to Cu 2+ chelates, which are eluted from the columns only with difficulty. 5 Human IFN-y is also strongly adsorbed to Cu z+ chelates, 6 but does not interact with most 1 L. Lundberg and J. Porfith, J. Chromatogr. 90, 87 (1975). 2 j. Porfith, J. Carlsson, I. Olsson, and G. Belfrage, Nature (London) 258, 598 (1975). 3 V. G. Edy, A. Billiau, and P. DeSomer, J. Biol. Chem. 252, 5934 (1977). 4 D. H. Coppenhaver, N. P. Sollenne, and B. H. Bowman, Arch. Biochem. Biophys. 226, 218 (1983). 5 K. C. Chada, P. M. Grob, A. J. Mikulski, L. R. Davis, Jr., and E. Sulkowski, J. Gen. Virol. 43, 701 (1979). 6 E. Sulkowski, K. Vastola, D. Oleszek, and W. von Meunchhausen, in "Affinity Chromatography and Related Techniques" (T. C. J. Gribnau, J. Visser, and R. J. F. Nivard, eds.), p. 313. Elsevier, Amsterdam, 1982.
METHODS IN ENZYMOLOGY.VOL. 119
Copyright© 1986by AcademicPress, Inc. All rightsof reproductionin any formreserved.
200
PURIFICATION OF INTERFERONS
[28]
other metal chelates, which Porath 2 has determined to be weaker adsorbents. 7 Human IFN-y is retained on iminodiacetic acid activated agarose columns which have been charged with Ni 2+, however. This moderately weak metal chelate gel shows an unexpected interaction with IFN-y, the mechanism of which has not been determined, which can be useful in its purification. Materials Sepharose 6B, Pharmacia Fine Chemicals 1,4-Butanediol diglycidylether, Aldrich Chem. Co. Iminodiacetic acid disodium salt, Aldrich Chem. Co. Sodium Borohydride, Sigma NiSO4" 7H20 (Gold Label), Aldrich Chem. Co. Tris(hydroxymethyl) aminomethane NazCO3 EDTA NaCI NaC2H302 NaHzPO4 Solutions 0.02 M Tris. HCI, 0.15 M NaC1, pH 8.5 (Buffer A) 0.1 M NaH2PO4, 0.15 M NaCI, pH 6.5 (Buffer B) 0.1 M NaCzH302, 1.0 M NaCI, pH 4.5 (Buffer C) 0.05 M EDTA, 1.0 M NaCI Interferon and Interferon Assay Human immune interferon was prepared from peripheral blood lymphocytes which had been stimulated with staphylococcal enterotoxin A (SEA) as previously described, s,9 Interferon was concentrated by chromatography on columns of silicic acid and eluted with 0.3 M (CH3)4NCI in PBS. J0 In some experiments, silicic acid concentrated interferon was further purified by gel filtration on Ultragel AcA54 eluted with 1.0 M NaC1 in PBS before Ni 2÷ chelate chromatography was performed. 1° Interferon 7D. H. Coppenhaver, unpublished observations. s M. P. Langford,J. A. Georgiades,G. J. Stanton, F. Dianzani, and H. M. Johnson, Infect. Immun. 26, 36 (1979). 9 H. M. Johnson, F. Dianzani, and J. A. Georgiades, this series, Vol. 78, p. 158. ~0V. M. Papermasterand S. Baron, Tex. Rep. Biol. Med. 41, 672 (1981-1982).
[28]
HUMAN IMMUNE INTERFERON
201
was assayed on human WISH cells by estimation of reduction of cytopathic effectU; interferon activity was expressed in terms of National Institutes of Health reference standard. P r e p a r a t i o n of I m i n o d i a c e t i c A c i d - A c t i v a t e d S e p h a r o s e an d Ni 2+ Chelate Column
Sepharose 6B is epoxy activated essentially as described by Porath.l,2 Swelled resin (150 ml) is dried in air (30 min on a sintered glass filter) before being added to a mixture of 100 ml 1,4-butanediol diglycidyl ether and 200 mg of NaBH4 in 100 ml of 0.5 M NaOH. The mixture is sealed and incubated with gentle agitation in a 25° water bath for 24 hr. The resin is then removed and exhaustively washed with 100-200 volumes of deionized water. The washed resin is suction dried on a sintered glass filter before being added to 20 g of iminodiacetic acid, disodium salt, dissolved in 100 ml of 2 M Na2CO3. The resin/iminodiacetic acid mixture is then placed in a 65 ° water bath and incubated with constant gentle agitation for 24 hr. After incubation, the iminodiacetic acid activated Sepharose 6B is exhaustively washed (100-200 volumes) with deionized water after which it is ready for chromatographic use. Columns of epoxy activated Sepharose 6B which have not been previously used are flushed with 0.05 M EDTA in 1.0 M NaCI to remove any metal ions which have been inadvertently bound to the resin. The EDTA is then flushed from the column with Buffer A before charging the resin w i t h N i 2+. The column is charged by passing a 2-3 mg/ml solution of NiSO4 in water or Buffer A through the column until the top 80% of the resin is completely saturated. The resin which has complexed with Ni 2+ will be pale blue; the uncomplexed resin serves as a reservoir to scavenge Ni 2+ ions which are displaced during the course of the chromatography. Finally, the column is reequilibrated with Buffer A before sample application. Nickel Chelate Chromatography Samples of partially purified immune interferon that have been dialyzed against Buffer A are loaded onto the column which is then developed at a linear flow rate of 10-15 ml/cm2/hr at 22°. The volume of the applied sample does not appear to be critical. The column is flushed with 4-5 column volumes of Buffer A, until unbound material is totally eluted. An intermediate pH buffer (Buffer B) may then be applied to the column u p. C. Familletti, S. Rubinstein, and S. Pestka, this series, Vol. 78, p. 387.
202
PURIFICATION OF INTERFERONS
[28]
QUANTITATIVE DATA ON NICKEL CHELATE CHROMATOGRAPHY OF HUMAN I F N - ' y
Sample Expt. A Silicic acid concentrated IFN-3, Eluate from Ni2+ column Peak fraction Expt. B AcA-54 purified IFN-'y Eluate from Ni-'+ column Peak fraction Expt. C AcA-54 purified IFN-~/ Eluate from Ni2+ column Peak fraction
Total units
Specific activity
Yield(%)
Purification factor
2.0 x 103
8.0 × 103
1.0 x 103
--
--
8.7
0.9 x l0~
7.8 × 103
7.8 x 104
98
78
2.9
2.0 x 103
5.8 x 103
2.0 x l0 s
72
200
9.2
3.0 x 103
2.7 × 104
4.3 x 103
--
--
2.5
X 10 3
3.0 X l04
1.7 X 10"
108
395
4.0
5.0 x 103
2.0 x 104
5.0 x 106
74
1,163
29.0
3.0 × 103
2.7 x 104
4.0 x 104
--
14.4
3.2 x 103
4.6 x 104
1.6 x l06
53
40
2.4
8.0 x l0 t
1.9 x 104
8.9 x 106
41
222
Volume (ml)
Units/ml
4.0
12.0
--
to r e m o v e w e a k l y a d s o r b e d m a t e r i a l . I n t e r f e r o n a c t i v i t y is r e c o v e r e d b y flushing with 4 - 5 c o l u m n v o l u m e s o f Buffer C. T h e e n t i r e c h r o m a t o g r a p h i c p r o c e d u r e m a y b e c a r r i e d o u t at 4°; h o w e v e r , slightly b e t t e r resol u t i o n a n d r e c o v e r y o f i n t e r f e r o n a c t i v i t y are o b t a i n e d w h e n the c o l u m n is m a i n t a i n e d at r o o m t e m p e r a t u r e . T h e c o l u m n effluent s h o u l d be m a i n t a i n e d at 4 ° to p r e s e r v e i n t e r f e r o n activity. T h e IFN-3, does n o t s e e m to be a d v e r s e l y affected b y s h o r t (24 hr) e x p o s u r e to the p H 4.5 buffer at 4 °. T o t a l r e c o v e r y o f a p p l i e d i n t e r f e r o n a c t i v i t y has r a n g e d f r o m 53 to 125% in v a r i o u s e x p e r i m e n t s ; g r e a t e r t h a n 90% r e c o v e r y is typical. F r o m 80 to 96% o f the r e c o v e r e d I F N - y a c t i v i t y is a s s o c i a t e d with the m a t e r i a l eluting at p H 4.5. T h e r e m a i n d e r is f o u n d in the initial w a s h . T y p i c a l r e s u l t s f r o m N i 2÷ c h e l a t e c h r o m a t o g r a p h y o f I F N - y p r e p a r a t i o n s are g i v e n in the table, a n d a r e p r e s e n t a t i v e c o l u m n profile is d e p i c t e d in Fig. 1.
Comments C h r o m a t o g r a p h y o n c o l u m n s o f e p o x y a c t i v a t e d a g a r o s e c h a r g e d with Ni 2÷ ions has p r o v e n to be a useful step in I F N - y purification. T h e tech-
[28]
HUMAN IMMUNE INTERFERON ,
~
,
203
10,000
o.15
E
tO
z 0.10
5000
to~
0 e.~
3
<
0.05
200 o.01 o
10
20
30
Fraction No.
FIG. 1. Nickel chelate chromatography of silicic acid concentrated human IFN-% A volume of 4.0 ml of concentrated, dialyzed IFN-3, was applied to a 1.5 × 12 cm column of iminodiacetic acid-activated Sepharose 6B which had been charged with Ni -'+ and equilibrated in 20 mM Tris. HCI, 0.15 M NaC1, pH 8.5. After 20 fractions of 2.9 ml had been collected, the elution buffer was changed to 0.1 M NaC2H302, 1.0 M NaCI, pH 4.5. Units of IFN activity (dashed line) and total UV absorbance (solid line) are shown. Quantitative data for this experiment are given in the table (Expt. A).
nique in general appears to be forgiving, in that useful results have been obtained under a variety of conditions. Experience has shown that some precautions will enhance the success of the technique. Although chromatography can be carried out under cold room conditions, total recovery of IFN-y activity is enhanced if the column is maintained at 22°. No deterioration of the separation resin has been observed after dozens of experiments, but results are enhanced if the column is stripped of bound metal ions with 50 mM EDTA and recharged with Ni 2÷ after each run. As might be expected theoretically, ~,2 maintenance of a basic pH (8.0-8.5) appears to be important to maximize the initial adsorption of the IFN-y to the Ni 2+ chelate column. Adsorption to metal chelates is effective at a wide range of ionic strengths, but appears to undergo a minimum at about 1 M NaCI. 12 Hence, an increased appearance of IFN activity in the wash fraction can be expected if the sample is loaded in buffers containing 1 M J2 j. Porftth and B. Olin,
Biochemistry 22, 1621 (1983).
204
PURIFICATION
OF INTERFERONS
[29]
NaCI or maintained at neutral pH. Sample volume, however, does not appear to be a critical variable. Although slightly better results have been obtained with more concentrated samples, even dilute preparations have been successfully chromatographed on these columns. In these cases (e.g., Expt. C, the table) a significant concentration of the IFN-y activity has been obtained, particularly in peak fractions. Thus, Ni 2+ chelate chromatography may be a useful technique for both purification and concentration of IFN-y.
[29] 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
Human Immune Interferon
B y H S I A N G - F U K U N G , Y U - C H I N G E . PAN, JOHN MOSCHERA, KELLY TSAI, EVA BEKESI, MAY CHANG, HIROMU SUGINO, a n d SusuMu HONDA
Full length cDNA coding for mature human immune interferon (IFNy) has been isolated and the coding sequence for mature IFN-y has been expressed in Escherichia coli, j-3 yeast, 4 and monkey cells. 1,2 We have purified recombinant immune interferon (IFN-y) produced in E. coli. The purification as well as structural characterization of the bacterial product is described here. Materials and General Procedures Bacteria and Bacterial Fermentation Escherichia coli RR1 (pRK248clts, pRC231/IFN-7) was used for fermentations to produce IFN-y. 3 pRC231/IFN-7 differs from pRC23/IFN3/3 in nucleotide spacing between the Shine-Dalgaro sequence and the ATG initiation codon, pRC2314a is a derivative of pBR322 containing the phage h PL promoter. Expression of the IFN-'y gene is controlled by the P. W. Gray, D. W. Leung, D. Pennica, E. Yelverton, R. Najarian, C. C. Simonsen, R. Derynck, P. J. Sherwood, D. M. Wallace, S. L. Berger, A. D. Levinson, and D. V. Goeddel, Nature (London)295, 503 (1982). 2 R. Devos, H. Cheroutre, Y. Taya, W. Degrave, H. van Heuverswyn, and W. Fiers, Nucleic Acids Res. 10, 2487 (1982). 3 R. Crowl, this volume [55]. 4 R. Derynck, A. Singh, and D. V. Goeddel, Nucleic Acids Res. U , 1819 (1983). 4~ R. Crowl et al. In preparation.
METHODS IN ENZYMOLOGY, VOL. 119
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