Journal of General Virology (1990), 71, 1057 1063. Printed in Great Britain
1057
Production, purification and biological properties of an Escherichia coil-derived recombinant porcine alpha interferon Francois Lef~vre,* Ren~ L'Haridon, Francisco Borras-Cuestat and Claude La Bonnardi~re Institut National de la Recherche Agronomique, Laboratoire de Virologie et Immunologie Mol~culaires, Centre de Recherches de Jouy-en-Josas, Domaine de Vilvert, 78350 Jouy-en-Josas, France
Recombinant plasmids for intracellular synthesis of mature porcine interferon alpha 1 (IFN-~I) in Escherichia coli were constructed. High amounts of antiviral activity were obtained [up to 4 x 10 s international units (IU) per ml of bacterial culture]. Recombinant porcine IFN-~I (rlFN-~I) was purified to homogeneity by monoclonal antibody immunoaffinity and was found to have the expected Mr (17- 5K) and N-terminal sequence (except for the apparent lack of an N-terminal methionine). Its specific antiviral activity was 5 x 10 7 to 1 0 x l 0 7 IU/mg MDBK cells. In vitro biological
properties of this purified rlFN-~I were compared to those of virus-induced porcine leukocyte interferon: the two interferons shared similar antigenic determinants and had the same ability to induce a cytocidal effect on primary cultures of pig kidney epithelial cells. However, rlFN-~I was at least six times more active in inducing an antiviral state on homologous porcine cells. These properties are discussed in the light of a possible in vivo use of the purified recombinant molecule.
Introduction
Recently we showed that, as in the human, bovine and equine species (Capon et al., 1985; Hauptmann & Swetly, 1985; Velan et al., 1985; Himmler et al., 1986), the porcine IFN-~ (PoIFN-~) multigene family can be divided into two homologous, but distinct classes of genes. The class I subfamily, located on porcine chromosome 1 (Yerle et al., 1986) contains at least 11 loci, of which nine have been cloned and two sequenced: PoIFN-~I and PoIFN-~2 (Lef6vre & La Bonnardi6re, 1986; F. Lef+vre, unpublished results). The class II subfamily, which appears to be tightly linked to the class I subfamily, contains at least six distinct loci (F. Lef6vre, unpublished results). The low level expression of the PoIFN-~I gene in Escherichia coli revealed that its potential product, a preprotein of 189 amino acids with a putative N-terminal signal peptide of 23 amino acids, had an IFN-~ biological activity (Lef6vre & La Bonnardi6re, 1986). in this article, we describe the construction of plasmids allowing the intracellular synthesis of large amounts of mature porcine recombinant IFN-~I (rIFN~1) in E. coli cells, its subsequent purification to homogeneity and some of its in vitro biological properties.
Since the discovery of interferon (IFN) in 1957 (Isaacs & Lindenmann, 1957), much evidence has been given on its involvement in the pathogenesis of and recovery from viral diseases, which strongly supports its use as an in vivo antiviral agent in man. Until recently, however, all studies showing a clear beneficial effect of endogenous or exogenous IFN in experimentally induced viral diseases were performed in a limited set of laboratory animal species. Extending the study of the IFN system to other species such as domestic animals should provide further information about its role in other viral disease models. With the help of the recent advent of recombinant DNA technology, IFN could also represent a new therapeutic agent with a broad antiviral spectrum and immunomodulatory properties in veterinary medicine, as recently shown in cattle (see Bielefeldt Ohmann et al., 1987, for a review). The pig represents an economically important animal species for food and hence the development of such a new therapeutic and/or prophylactic agent against infectious diseases in this species is strongly desirable. Moreover, the pig is an interesting model for investigating the role of endogenous IFN in viral infections and for studying the pharmacology and toxicology of exogenous homologous IFN (La Bonnardi6re et al., 1984). t Present address: Universidadde Navarra, Facultadde Medicina, Dpto de Medicina Interna, Apartado 273, Pamplona, Spain. 0000-9241 © 1990 SGM
Methods Bacterial strains, plasmids and growth conditions. Plasmid and bacteriophageM13constructionswereclonedin E. coli strain RR1 and JM103, respectively.For experimentsin recombinantIFN-c~expres-
1058
F. Lefbvre and others PheAlaSerCysLeu 5'-TTCGCCAGCTGCCTC
cro
Pvull ~ J !al'N"T I"I"~gm
]-
3'
Shine-Dalgarno
sequence [ Xbal
ProSnd
1 Sa I
Gro o al' -p g
5' GCCTTAAGATCTGTTAACO~AGGAGG~CTAGATGGATCCGGCCGTCG--AC
Xbal
symheuc double-stranded DNA fragment
LedLeuSerCysAsn 5 ' -CTACTCAGCTGCAAT-3
Met CysAspLe uPro 5 '-TCTCATATGTGTGACC" ~CC2 3'
' Hpal
[
PvulI
Site-directed ~ mutagenesism_
Sail
~i~
--[---
Nde I
PvulI, HpaI ] isolate 675 bp fragment
Xbal
PolIK + 4 dN'lPs SalI
] PoI|K +dTTP [ Mung bean nucleasc / s~n I~Iate 0.65 kb fragment
T4 DNA ligase
T4 DNA lJgase cro- [3gal'-IFN ~xl
cro- B eal' ~ e t l F N _ 0 t ~ ~
p
[
pLD67
~
I p
cro [~gal' "
PolIK + 4 dNTPs m .,.iSmal Hmdlll J Isolmelarge fragment •
MetlFN0tl
~i~ii::~ ~ - ] t / l ..........
]
SmaI
Fig. 1. Construction of plasmids allowing the expression of porcine rlFN-cd. For more details, see Methods. Blackened box and open box represent the thermosensitive repressor gene (c/857) of the bacteriophage 2 and the ampicillin resistance gene, respectively. Sequences encoding the PolFN-cd signal peptide and mature protein are indicated by stippled and light grey boxes, respectively. The dark grey box represents the cro-flgal' coding sequence and the hatched box the Shine-Dalgarno sequence. The black arrow indicates the rightward promoter (PR) of bacteriophage lambda. Thin lines mark pBR322 or bacteriophage 2 sequences; bold lines show the porcine genomic non-coding sequences. PollK, the Klenow fragment of E. coli DNA polymerase I.
sion we used the E. coli protease-deficient (Ion-) strain CAGl139 (Grossman et al., 1983) transformed with the appropriate expression vector and grown in the enriched medium described by Mott et al. (1985) containing 3-2~ bactotryptone, 2 ~ yeast extract, 0.5~ Na2HPO4, 0.3% KH2PO4, 0.1% NH,,C1, 0-05% NaC1, 0.1 mMMgSO4, 0.001 mM-FeC13 in the presence of ampicillin (100 to 150 ~tg/ml). DNA manipulation, plasmid constructions and nucleotide sequence analysis. All enzymes were used according to the manufacturer's recommendations. Oligonucleotides were synthesized on a Biosearch 8600 DNA synthesizer and purified further as described (Sambrook et al., 1989). DNA manipulations and plasmid constructions were done according to standard procedures (Sambrook et al., 1989) and are summarized in Fig. 1. Plasmid pD1A9225 (generously provided by Dr Antoine Danchin, Institut Pasteur, Paris, France) was the basic vector used for porcine 1FN-cd expression. It allowed thermoinducible expression of heterologous coding sequences fused to the C terminus of a cr~fl-galactosidase peptide (cr~flgal', 49 amino acids) (Leplatois & Danchin, 1983). Plasmid pD1APoIFN-cd was constructed by inserting, at the unique PvuII site of pD1 A9225, the PvuII-Hpal fragment of the PoIFN-cd gene (containing sequences encoding part of the signal peptide and the complete mature protein) in the orientation allowing in-frame fusion with the cro-flgal' coding sequence. Plasmid pLD67 was constructed by cloning in the proper orientation (Lathe et al., 1984)
a 49 bp synthetic blunt-ended DNA fragment at the PvulI site of pD1A9225. Due to this insert, translation of the cro-flgal' sequence stopped at an in-frame TAA codon which was followed, 13bp downstream, by the consensus Shine-Dalgarno sequence 5' AAGG A G G T 3' (Gold, 1988) leading to a translational (re)initiation at a coding region inserted in the neighbouring unique XbaI site. Oligonucleotide-directed mutagenesis in bacteriophage M 13 (Zoller & Smith, 1982) was used to construct a gene encoding methionyl IFN-~I (MetIFN-~I): the sequence 5' CATATG 3' containing a methionine initiator codon and a unique NdeI restriction site was introduced just before the first codon (TGT, cysteine) of the putative mature IFN-cd coding sequence. Plasmid pLD67MetIFN-~l was constructed by inserting the MetIFN-cd coding sequence between the XbaI and SalI sites of pLD67 as described in Fig. 1. The nucleotide sequence extending between the cro-flgal' and MetlFN-~I coding regions was controlled according to Chen & Seeburg (1985) using a synthetic DNA primer. Monoclonal antibodies (MAbs). A peptide corresponding to the 15 Cterminal residues of PolFN-cd (Lef6vre & La Bonnardi~re, 1986) was synthesized by the method of Merrifield using the Fmoc-polyamide mode (Atherton & Sheppard, 1985) and coupled to ovalbumin via glutaraldehyde. Fifty ~tg of conjugate was injected twice into 3 month old BALB/c mice with a 20 day interval. Boosted splenocytes were fused with selectable murine SP2/O myeloma cells and hybridomas
Recombinant porcine IFN-~
were selected in medium containing azaserine (Galfre & Milstein, 1981). Hybridomas were screened using a direct ELISA in plastic plates coated with the conjugate. Five MAbs which recognized both the synthetic peptide and the denatured form of rlFN-~I (not shown) were isolated. One of them, C8-18, was used for immunoblotting experiments. Other MAbs were prepared against the complete rlFN-~I molecule. Two month old BALB/c mice were immunized over a 130 day period by four injections of porcine IFN [105 international units (IU), half intraperitoneal and half intravenous] one of which consisted of porcine leukocyte IFN (PolFN-Le) and the others of the crude rIFN-cd preparation described below. Fusion between splenocytes and SP2/O cells was performed 4 days after the last injection, and growing hybridomas were screened for IFN reactivity by an immunosorbent bioassay set up in our laboratory (R. L'Haridon et.aL, unpublished results). Eight independent anti-IFN-c~ MAbs were isolated; one of them, C22, was used for the immunoaffinity purification of rIFN-cd.
Purification of rIFN-cG. E. coli strain CAGlI39 harbouring the plasmid pLD67MetlFN-cdAHS was grown overnight at 37 °C in enriched medium to an optical density at 600 nm (OD6o0) of about 5. Extraction of rlFN-ctl from the bacterial pellet was conducted essentially as described by Valenzuela et al. (1985). The crude soluble extract was dialysed extensively against phosphate-buffered saline containing 0.02mM-PMSF, and rlFN-ctl was purified from this dialysate by a single-step immunoaffinity using specific anti-porcine rlFN-ctl MAb C22: monoclonal IgG was coupled to glutaraldehydeactivated amino-hexyl-Sepharose (Pharmacia) according to Cambiaso et al. (1975). Bound IFN was eluted from the column with 0.1 M-acetic acid, 0.3 M-NaC1, pH 2.5, extensively dialysed as above and analysed by SDS-PAGE and reverse-phase HPLC. About 200 pmol of protein was subjected to N-terminal amino acid sequencing by sequential Edman degradation using a gas-phase Applied Biosystem 470A apparatus, equipped with on-line phenylthiohydantoin (PTH) amino acid analyser 120A. Immunoblotting. Total bacterial proteins (0.5 OD600 unit of cell culture per gel slot) were separated by SDS-PAGE (Laemmli, 1970) followed by transfer to a nitrocellulose filter (Towbin et al., 1979). Immunodetection of rlFN-~I was performed using a crude ascitic fluid containing MAb C8-18; the bound antibody was revealed using 35Slabelled Protein A (Amersham, 875 Ci/mmol, 100 gCi/ml) according to Burnette (1981), and autoradiography of the filter. IFN-ct antiviral assay and neutralization. E. coli cell extracts were prepared for an IFN-ct antiviral assay by resuspending the bacterial pellet from 1 ml of culture in 200 gl of TNE buffer (10 mM-Tris-HCl pH 8.0, 1 mM-EDTA, 100mM-NaC1), mixing the suspension with 200gl of lysis solution (10M-urea, 2% w/v SDS, 2% v/v 2mercaptoethanol) and heating the lysate for 1 min at 100 °C. The IFN antiviral activity was assayed by inhibition of the c.p.e, of vesicular stomatitis virus on MDBK or other cell lines, as previously described (La Bonnardi6re & Laude, 1981). Antiviral titres were expressed in IU by comparison with the international human IFN-Le (HulFN-Le) reference Ga23-902-530 (NIH, Bethesda, Md., U.S.A.). Neutralization of IFN antiviral activity was performed with a sheep anti-human IFNct serum using a previously described constant antibody method (La Bonnardidre et al., 1986).
Results High level expression o f porcine rlFN-~I in E. coli cells T o test its a b i l i t y for b e i n g e x p r e s s e d at a h i g h level in E. coli, the P o I F N - ~ I c o d i n g sequence was i n s e r t e d into the
1059
fused p r o t e i n e x p r e s s i o n p l a s m i d p D 1 A 9 2 2 5 . T h e plasm i d o b t a i n e d , t e r m e d p D 1 A P o l F N - ~ I , p o t e n t i a l l y codes for a 224 a m i n o a c i d h y b r i d p r o t e i n c o n s i s t i n g o f the entire m a t u r e I F N - ~ I a n d r e m a i n i n g nine a m i n o acids o f the signal p e p t i d e (S15 to $23) fused at the N t e r m i n u s to the 49 residues o f cro-flgal'. T h e p D 1 A P o l F N - ~ I c o n s t r u c t was e x a m i n e d for p r o t e i n synthesis in the protease-deficient strain (lon-) E. coli C A G 1139. L a r g e a m o u n t s o f a p r o t e i n species w i t h the e x p e c t e d Mr (28K) was d e t e c t e d by S D S - P A G E o f total E. coli p r o t e i n s after h e a t i n d u c t i o n (Fig. 2a). W e s t e r n blot analysis o f the gel s h o w e d t h a t a b a n d o f s i m i l a r size was the m a j o r p r o t e i n species i m m u n o r e a c t i v e w i t h the M A b C8-18 (Fig. 2b). A s the a i m o f this w o r k was to o b t a i n a r e c o m b i n a n t m a t u r e P o l F N - c d d e v o i d o f its signal p e p t i d e , we chose to express the M e t l F N - ~ I p r o t e i n ( m a t u r e I F N - ~ I w i t h an a d d i t i o n a l N - t e r m i n a l m e t h i o n i n e ) in E. coli. A s p D 1 A 9 2 2 5 s e e m e d to express the P o l F N - ~ I c o d i n g sequence efficiently, we m o d i f i e d it to o b t a i n p L D 6 7 w h i c h p e r m i t t e d the e x p r e s s i o n of u n f u s e d c o d i n g sequences. T h e sequence e n c o d i n g M e t l F N - ~ I was c o n s t r u c t e d by site-directed m u t a g e n e s i s o f the p r e l F N ~1 c o d i n g sequence a n d inserted into p L D 6 7 . T h e plasmid obtained, pLD67MetlFN-~l, e n a b l e d the e x p r e s s i o n o f a c r o - f l g a l ' p e p t i d e (of 51 residues) a n d M e t I F N - ~ I f r o m a b i c i s t r o n i c m R N A molecule. E. coli C A G 1 1 3 9 cells b e a r i n g this construct grown a n d heati n d u c e d as d e s c r i b e d in Fig. 2(c) c o n t a i n e d a h i g h a n t i v i r a l a c t i v i t y (1.2 x 10 s I U / m l o f b a c t e r i a l culture on M D B K cells). In o r d e r to o b t a i n constitutive expression o f the r e c o m b i n a n t protein, the H i n d I I I - S m a I f r a g m e n t b e a r i n g the Y e n d o f the ci857 t h e r m o s e n s i t i v e r e p r e s s o r gene was deleted from p L D 6 7 M e t I F N - e l (Fig. 1). A s a t u r a t i o n culture o f E. coli C A G 1 1 3 9 h a r b o u r i n g this new c o n s t r u c t i o n ( p L D 6 7 M e t I F N - c d A H S ) c o n t a i n e d at least three times m o r e a n t i v i r a l a c t i v i t y (4 x 10 s I U / m l o f b a c t e r i a l culture on M D B K cells) t h a n the p r e v i o u s one. W e s t e r n blot analysis p e r f o r m e d for b o t h constructs r e v e a l e d t h a t a single p r o t e i n species w i t h an a p p a r e n t Mr o f 17.5K c o m i g r a t i n g w i t h IFN-c~ a n t i v i r a l a c t i v i t y a n d c o r r e s p o n d i n g to m a t u r e p o r c i n e r I F N - c d was i m m u n o d e t e c t e d in E. coli cell extracts by the C8-18 M A b (Fig. 2c).
Purification and N-term&at sequencing o f mature porcine rlFN-cd Biologically active r l F N - c d was e x t r a c t e d a n d purified as d e s c r i b e d in M e t h o d s . S D S - P A G E analysis r e v e a l e d a single b a n d w i t h a n Mr o f 17"5K after C o o m a s s i e blue staining (Fig. 2d). A t this stage, specific a c t i v i t y o f the purified r l F N - e l r a n g e d a r o u n d 5 x 107 I U p e r m g o f p r o t e i n on M D B K cells. N - t e r m i n a l s e q u e n c i n g c o n f i r m e d t h a t the r e c o m b i n a n t I F N was the m a t u r e form. A s the p r o t e i n
F. LeJbvre and others
1060
(a)
(b) 1
2
1
=
2
"
,
"
(d)
(c) 1
.....
2
1
3
2
200K~
-"
92.5K~ 69K
46K
30K
20.1K~
-
....
-
21.5K~
..... 14.4K~
':" :~'
g
!
f
_/:
'
:
.....
Fig. 2. Characterization (by SDS P A G E and Western blot analysis) and purification of porcine IFN-ct 1 expressed in E . c o l i C A G 11399 cells harbouring various expression plasmids. (a) Cells bearing plasmid pD1APolFN-ctl were grown at a restrictive temperature (30 °C) in the enriched medium described in Methods to an OD6o o of 1-5. The culture was maintained at the same temperature (lane 1) or at an induction temperature (42 °C, lane 2) for 2 h. Proteins were analysed on a 12.5~ SDS polyacrylamide gel and stained with Coomassie blue. (b) Western blot analysis of the gel in (a) using the C8-18 MAb. Lanes 1 and 2 refer to homologous samples of the gel in (a). The arrow indicates the position of the 28K cr~flgal' IFN-cd fusion protein. Other minor bands probably correspond to degradation products. (c) Cells bearing plasmid pLD67MetlFN-cd were grown and induced as in (a) except that induction was performed at an OD600 of 2. Lane l, non-induced culture; lane 2, induced culture. Cells bearing pLD67MetIFN-cdAHS were grown overnight at 37 °C in the same medium (lane 3). Bacterial proteins were separated on a 15~ SDS-polyacrylamide gel and Western blot analysis was performed as in (a). (d) Purification of mature porcine rlFN-cd. Proteins were analysed on a 15~ SDS polyacrylamide gel and stained with Coomassie blue. Lane 1, total proteins from cells harbouring plasmid pLD67MetlFN-cdAHS; lane 2, approximately 2 gg of immunoaffinity-purified porcine rlFN-~I. Mr standards are indicated on the left of (a), (b), (c) and (d).
subjected to the Edman degradation was not reduced or alkylated, the first N-terminal cysteine residue did not give a P T H derivative. We thus obtained: none-D-L-PQ-T-H-S-L-A in the first 10 steps which clearly corresponds to the terminal sequence deduced from the D N A (Lef~vre & La Bonnardi6re, 1986). On account of the yield of the first step of the Edman degradation (our result was 27~), which was within the normal range of yields (20 to 50~), and as no methionine-PTH was found, we think that at least a major fraction of rIFN-cd was non-methionylated and unblocked at its N terminus; however we cannot exclude the existence of a minor fraction, methionylated and blocked at its N terminus.
In vitro biological properties of rlFN-aI (i) Antiviral effect on different cells The antiviral spectrum of rIFN-~I was compared with that of PoIFN-Le obtained by infection of pig peripheral blood lymphocytes with influenza virus (La Bonnardi6re et al., 1986). For that purpose, we used a panel of five cell lines belonging to four different species (Table l). It appeared that, like IFN-Le, rIFN-al exhibited a broad spectrum of antiviral activity ( 1 0 ~ activity on mouse cells as compared to M D B K cells). But interestingly, rlFN-~I was clearly more active (by sixfold) than its natural counterpart on homologous porcine cells. This
Recombinant porcine IFN-~
Table 1. Antiviral activity of porcine IFN-Le and rlFN-cd on cell lines fi'om different species*
Table 3. Neutralization of porc&e IFN antiviral activity* by an anti-human IFN-~ serum
Antiviral activity (IU/ml) Species
Cell line
IFN-Le
rlFN-~I
Bovine Human Murine Porcine
MDBK WISH L929 PD5 ST83
3000 330 440 330 110
3000 330 250 2200 660
* The antiviral titres of porcine IFN-Le and rlFN-c~l preparations having the same titre on MDBK cells (3000 IU/ml) were determined also on human, murine and porcine cell lines.
Table 2. Antiviral and cytocidal activities of porcine and human IFNs on low-passage porcine kidney cells (RPa 5495)* Cells MDBK IFN PolFN-Le rlFN-cd HulFN-Le
Anti-human IFN-~ serumS" + + +
RPa 5495
AVA
AVA
CCA
8 2 10 4.5 8.5 1
7
1057
Production, purification and biological properties of an Escherichia coil-derived recombinant porcine alpha interferon Francois Lef~vre,* Ren~ L'Haridon, Francisco Borras-Cuestat and Claude La Bonnardi~re Institut National de la Recherche Agronomique, Laboratoire de Virologie et Immunologie Mol~culaires, Centre de Recherches de Jouy-en-Josas, Domaine de Vilvert, 78350 Jouy-en-Josas, France
Recombinant plasmids for intracellular synthesis of mature porcine interferon alpha 1 (IFN-~I) in Escherichia coli were constructed. High amounts of antiviral activity were obtained [up to 4 x 10 s international units (IU) per ml of bacterial culture]. Recombinant porcine IFN-~I (rlFN-~I) was purified to homogeneity by monoclonal antibody immunoaffinity and was found to have the expected Mr (17- 5K) and N-terminal sequence (except for the apparent lack of an N-terminal methionine). Its specific antiviral activity was 5 x 10 7 to 1 0 x l 0 7 IU/mg MDBK cells. In vitro biological
properties of this purified rlFN-~I were compared to those of virus-induced porcine leukocyte interferon: the two interferons shared similar antigenic determinants and had the same ability to induce a cytocidal effect on primary cultures of pig kidney epithelial cells. However, rlFN-~I was at least six times more active in inducing an antiviral state on homologous porcine cells. These properties are discussed in the light of a possible in vivo use of the purified recombinant molecule.
Introduction
Recently we showed that, as in the human, bovine and equine species (Capon et al., 1985; Hauptmann & Swetly, 1985; Velan et al., 1985; Himmler et al., 1986), the porcine IFN-~ (PoIFN-~) multigene family can be divided into two homologous, but distinct classes of genes. The class I subfamily, located on porcine chromosome 1 (Yerle et al., 1986) contains at least 11 loci, of which nine have been cloned and two sequenced: PoIFN-~I and PoIFN-~2 (Lef6vre & La Bonnardi6re, 1986; F. Lef+vre, unpublished results). The class II subfamily, which appears to be tightly linked to the class I subfamily, contains at least six distinct loci (F. Lef6vre, unpublished results). The low level expression of the PoIFN-~I gene in Escherichia coli revealed that its potential product, a preprotein of 189 amino acids with a putative N-terminal signal peptide of 23 amino acids, had an IFN-~ biological activity (Lef6vre & La Bonnardi6re, 1986). in this article, we describe the construction of plasmids allowing the intracellular synthesis of large amounts of mature porcine recombinant IFN-~I (rIFN~1) in E. coli cells, its subsequent purification to homogeneity and some of its in vitro biological properties.
Since the discovery of interferon (IFN) in 1957 (Isaacs & Lindenmann, 1957), much evidence has been given on its involvement in the pathogenesis of and recovery from viral diseases, which strongly supports its use as an in vivo antiviral agent in man. Until recently, however, all studies showing a clear beneficial effect of endogenous or exogenous IFN in experimentally induced viral diseases were performed in a limited set of laboratory animal species. Extending the study of the IFN system to other species such as domestic animals should provide further information about its role in other viral disease models. With the help of the recent advent of recombinant DNA technology, IFN could also represent a new therapeutic agent with a broad antiviral spectrum and immunomodulatory properties in veterinary medicine, as recently shown in cattle (see Bielefeldt Ohmann et al., 1987, for a review). The pig represents an economically important animal species for food and hence the development of such a new therapeutic and/or prophylactic agent against infectious diseases in this species is strongly desirable. Moreover, the pig is an interesting model for investigating the role of endogenous IFN in viral infections and for studying the pharmacology and toxicology of exogenous homologous IFN (La Bonnardi6re et al., 1984). t Present address: Universidadde Navarra, Facultadde Medicina, Dpto de Medicina Interna, Apartado 273, Pamplona, Spain. 0000-9241 © 1990 SGM
Methods Bacterial strains, plasmids and growth conditions. Plasmid and bacteriophageM13constructionswereclonedin E. coli strain RR1 and JM103, respectively.For experimentsin recombinantIFN-c~expres-
1058
F. Lefbvre and others PheAlaSerCysLeu 5'-TTCGCCAGCTGCCTC
cro
Pvull ~ J !al'N"T I"I"~gm
]-
3'
Shine-Dalgarno
sequence [ Xbal
ProSnd
1 Sa I
Gro o al' -p g
5' GCCTTAAGATCTGTTAACO~AGGAGG~CTAGATGGATCCGGCCGTCG--AC
Xbal
symheuc double-stranded DNA fragment
LedLeuSerCysAsn 5 ' -CTACTCAGCTGCAAT-3
Met CysAspLe uPro 5 '-TCTCATATGTGTGACC" ~CC2 3'
' Hpal
[
PvulI
Site-directed ~ mutagenesism_
Sail
~i~
--[---
Nde I
PvulI, HpaI ] isolate 675 bp fragment
Xbal
PolIK + 4 dN'lPs SalI
] PoI|K +dTTP [ Mung bean nucleasc / s~n I~Iate 0.65 kb fragment
T4 DNA ligase
T4 DNA lJgase cro- [3gal'-IFN ~xl
cro- B eal' ~ e t l F N _ 0 t ~ ~
p
[
pLD67
~
I p
cro [~gal' "
PolIK + 4 dNTPs m .,.iSmal Hmdlll J Isolmelarge fragment •
MetlFN0tl
~i~ii::~ ~ - ] t / l ..........
]
SmaI
Fig. 1. Construction of plasmids allowing the expression of porcine rlFN-cd. For more details, see Methods. Blackened box and open box represent the thermosensitive repressor gene (c/857) of the bacteriophage 2 and the ampicillin resistance gene, respectively. Sequences encoding the PolFN-cd signal peptide and mature protein are indicated by stippled and light grey boxes, respectively. The dark grey box represents the cro-flgal' coding sequence and the hatched box the Shine-Dalgarno sequence. The black arrow indicates the rightward promoter (PR) of bacteriophage lambda. Thin lines mark pBR322 or bacteriophage 2 sequences; bold lines show the porcine genomic non-coding sequences. PollK, the Klenow fragment of E. coli DNA polymerase I.
sion we used the E. coli protease-deficient (Ion-) strain CAGl139 (Grossman et al., 1983) transformed with the appropriate expression vector and grown in the enriched medium described by Mott et al. (1985) containing 3-2~ bactotryptone, 2 ~ yeast extract, 0.5~ Na2HPO4, 0.3% KH2PO4, 0.1% NH,,C1, 0-05% NaC1, 0.1 mMMgSO4, 0.001 mM-FeC13 in the presence of ampicillin (100 to 150 ~tg/ml). DNA manipulation, plasmid constructions and nucleotide sequence analysis. All enzymes were used according to the manufacturer's recommendations. Oligonucleotides were synthesized on a Biosearch 8600 DNA synthesizer and purified further as described (Sambrook et al., 1989). DNA manipulations and plasmid constructions were done according to standard procedures (Sambrook et al., 1989) and are summarized in Fig. 1. Plasmid pD1A9225 (generously provided by Dr Antoine Danchin, Institut Pasteur, Paris, France) was the basic vector used for porcine 1FN-cd expression. It allowed thermoinducible expression of heterologous coding sequences fused to the C terminus of a cr~fl-galactosidase peptide (cr~flgal', 49 amino acids) (Leplatois & Danchin, 1983). Plasmid pD1APoIFN-cd was constructed by inserting, at the unique PvuII site of pD1 A9225, the PvuII-Hpal fragment of the PoIFN-cd gene (containing sequences encoding part of the signal peptide and the complete mature protein) in the orientation allowing in-frame fusion with the cro-flgal' coding sequence. Plasmid pLD67 was constructed by cloning in the proper orientation (Lathe et al., 1984)
a 49 bp synthetic blunt-ended DNA fragment at the PvulI site of pD1A9225. Due to this insert, translation of the cro-flgal' sequence stopped at an in-frame TAA codon which was followed, 13bp downstream, by the consensus Shine-Dalgarno sequence 5' AAGG A G G T 3' (Gold, 1988) leading to a translational (re)initiation at a coding region inserted in the neighbouring unique XbaI site. Oligonucleotide-directed mutagenesis in bacteriophage M 13 (Zoller & Smith, 1982) was used to construct a gene encoding methionyl IFN-~I (MetIFN-~I): the sequence 5' CATATG 3' containing a methionine initiator codon and a unique NdeI restriction site was introduced just before the first codon (TGT, cysteine) of the putative mature IFN-cd coding sequence. Plasmid pLD67MetIFN-~l was constructed by inserting the MetIFN-cd coding sequence between the XbaI and SalI sites of pLD67 as described in Fig. 1. The nucleotide sequence extending between the cro-flgal' and MetlFN-~I coding regions was controlled according to Chen & Seeburg (1985) using a synthetic DNA primer. Monoclonal antibodies (MAbs). A peptide corresponding to the 15 Cterminal residues of PolFN-cd (Lef6vre & La Bonnardi~re, 1986) was synthesized by the method of Merrifield using the Fmoc-polyamide mode (Atherton & Sheppard, 1985) and coupled to ovalbumin via glutaraldehyde. Fifty ~tg of conjugate was injected twice into 3 month old BALB/c mice with a 20 day interval. Boosted splenocytes were fused with selectable murine SP2/O myeloma cells and hybridomas
Recombinant porcine IFN-~
were selected in medium containing azaserine (Galfre & Milstein, 1981). Hybridomas were screened using a direct ELISA in plastic plates coated with the conjugate. Five MAbs which recognized both the synthetic peptide and the denatured form of rlFN-~I (not shown) were isolated. One of them, C8-18, was used for immunoblotting experiments. Other MAbs were prepared against the complete rlFN-~I molecule. Two month old BALB/c mice were immunized over a 130 day period by four injections of porcine IFN [105 international units (IU), half intraperitoneal and half intravenous] one of which consisted of porcine leukocyte IFN (PolFN-Le) and the others of the crude rIFN-cd preparation described below. Fusion between splenocytes and SP2/O cells was performed 4 days after the last injection, and growing hybridomas were screened for IFN reactivity by an immunosorbent bioassay set up in our laboratory (R. L'Haridon et.aL, unpublished results). Eight independent anti-IFN-c~ MAbs were isolated; one of them, C22, was used for the immunoaffinity purification of rIFN-cd.
Purification of rIFN-cG. E. coli strain CAGlI39 harbouring the plasmid pLD67MetlFN-cdAHS was grown overnight at 37 °C in enriched medium to an optical density at 600 nm (OD6o0) of about 5. Extraction of rlFN-ctl from the bacterial pellet was conducted essentially as described by Valenzuela et al. (1985). The crude soluble extract was dialysed extensively against phosphate-buffered saline containing 0.02mM-PMSF, and rlFN-ctl was purified from this dialysate by a single-step immunoaffinity using specific anti-porcine rlFN-ctl MAb C22: monoclonal IgG was coupled to glutaraldehydeactivated amino-hexyl-Sepharose (Pharmacia) according to Cambiaso et al. (1975). Bound IFN was eluted from the column with 0.1 M-acetic acid, 0.3 M-NaC1, pH 2.5, extensively dialysed as above and analysed by SDS-PAGE and reverse-phase HPLC. About 200 pmol of protein was subjected to N-terminal amino acid sequencing by sequential Edman degradation using a gas-phase Applied Biosystem 470A apparatus, equipped with on-line phenylthiohydantoin (PTH) amino acid analyser 120A. Immunoblotting. Total bacterial proteins (0.5 OD600 unit of cell culture per gel slot) were separated by SDS-PAGE (Laemmli, 1970) followed by transfer to a nitrocellulose filter (Towbin et al., 1979). Immunodetection of rlFN-~I was performed using a crude ascitic fluid containing MAb C8-18; the bound antibody was revealed using 35Slabelled Protein A (Amersham, 875 Ci/mmol, 100 gCi/ml) according to Burnette (1981), and autoradiography of the filter. IFN-ct antiviral assay and neutralization. E. coli cell extracts were prepared for an IFN-ct antiviral assay by resuspending the bacterial pellet from 1 ml of culture in 200 gl of TNE buffer (10 mM-Tris-HCl pH 8.0, 1 mM-EDTA, 100mM-NaC1), mixing the suspension with 200gl of lysis solution (10M-urea, 2% w/v SDS, 2% v/v 2mercaptoethanol) and heating the lysate for 1 min at 100 °C. The IFN antiviral activity was assayed by inhibition of the c.p.e, of vesicular stomatitis virus on MDBK or other cell lines, as previously described (La Bonnardi6re & Laude, 1981). Antiviral titres were expressed in IU by comparison with the international human IFN-Le (HulFN-Le) reference Ga23-902-530 (NIH, Bethesda, Md., U.S.A.). Neutralization of IFN antiviral activity was performed with a sheep anti-human IFNct serum using a previously described constant antibody method (La Bonnardidre et al., 1986).
Results High level expression o f porcine rlFN-~I in E. coli cells T o test its a b i l i t y for b e i n g e x p r e s s e d at a h i g h level in E. coli, the P o I F N - ~ I c o d i n g sequence was i n s e r t e d into the
1059
fused p r o t e i n e x p r e s s i o n p l a s m i d p D 1 A 9 2 2 5 . T h e plasm i d o b t a i n e d , t e r m e d p D 1 A P o l F N - ~ I , p o t e n t i a l l y codes for a 224 a m i n o a c i d h y b r i d p r o t e i n c o n s i s t i n g o f the entire m a t u r e I F N - ~ I a n d r e m a i n i n g nine a m i n o acids o f the signal p e p t i d e (S15 to $23) fused at the N t e r m i n u s to the 49 residues o f cro-flgal'. T h e p D 1 A P o l F N - ~ I c o n s t r u c t was e x a m i n e d for p r o t e i n synthesis in the protease-deficient strain (lon-) E. coli C A G 1139. L a r g e a m o u n t s o f a p r o t e i n species w i t h the e x p e c t e d Mr (28K) was d e t e c t e d by S D S - P A G E o f total E. coli p r o t e i n s after h e a t i n d u c t i o n (Fig. 2a). W e s t e r n blot analysis o f the gel s h o w e d t h a t a b a n d o f s i m i l a r size was the m a j o r p r o t e i n species i m m u n o r e a c t i v e w i t h the M A b C8-18 (Fig. 2b). A s the a i m o f this w o r k was to o b t a i n a r e c o m b i n a n t m a t u r e P o l F N - c d d e v o i d o f its signal p e p t i d e , we chose to express the M e t l F N - ~ I p r o t e i n ( m a t u r e I F N - ~ I w i t h an a d d i t i o n a l N - t e r m i n a l m e t h i o n i n e ) in E. coli. A s p D 1 A 9 2 2 5 s e e m e d to express the P o l F N - ~ I c o d i n g sequence efficiently, we m o d i f i e d it to o b t a i n p L D 6 7 w h i c h p e r m i t t e d the e x p r e s s i o n of u n f u s e d c o d i n g sequences. T h e sequence e n c o d i n g M e t l F N - ~ I was c o n s t r u c t e d by site-directed m u t a g e n e s i s o f the p r e l F N ~1 c o d i n g sequence a n d inserted into p L D 6 7 . T h e plasmid obtained, pLD67MetlFN-~l, e n a b l e d the e x p r e s s i o n o f a c r o - f l g a l ' p e p t i d e (of 51 residues) a n d M e t I F N - ~ I f r o m a b i c i s t r o n i c m R N A molecule. E. coli C A G 1 1 3 9 cells b e a r i n g this construct grown a n d heati n d u c e d as d e s c r i b e d in Fig. 2(c) c o n t a i n e d a h i g h a n t i v i r a l a c t i v i t y (1.2 x 10 s I U / m l o f b a c t e r i a l culture on M D B K cells). In o r d e r to o b t a i n constitutive expression o f the r e c o m b i n a n t protein, the H i n d I I I - S m a I f r a g m e n t b e a r i n g the Y e n d o f the ci857 t h e r m o s e n s i t i v e r e p r e s s o r gene was deleted from p L D 6 7 M e t I F N - e l (Fig. 1). A s a t u r a t i o n culture o f E. coli C A G 1 1 3 9 h a r b o u r i n g this new c o n s t r u c t i o n ( p L D 6 7 M e t I F N - c d A H S ) c o n t a i n e d at least three times m o r e a n t i v i r a l a c t i v i t y (4 x 10 s I U / m l o f b a c t e r i a l culture on M D B K cells) t h a n the p r e v i o u s one. W e s t e r n blot analysis p e r f o r m e d for b o t h constructs r e v e a l e d t h a t a single p r o t e i n species w i t h an a p p a r e n t Mr o f 17.5K c o m i g r a t i n g w i t h IFN-c~ a n t i v i r a l a c t i v i t y a n d c o r r e s p o n d i n g to m a t u r e p o r c i n e r I F N - c d was i m m u n o d e t e c t e d in E. coli cell extracts by the C8-18 M A b (Fig. 2c).
Purification and N-term&at sequencing o f mature porcine rlFN-cd Biologically active r l F N - c d was e x t r a c t e d a n d purified as d e s c r i b e d in M e t h o d s . S D S - P A G E analysis r e v e a l e d a single b a n d w i t h a n Mr o f 17"5K after C o o m a s s i e blue staining (Fig. 2d). A t this stage, specific a c t i v i t y o f the purified r l F N - e l r a n g e d a r o u n d 5 x 107 I U p e r m g o f p r o t e i n on M D B K cells. N - t e r m i n a l s e q u e n c i n g c o n f i r m e d t h a t the r e c o m b i n a n t I F N was the m a t u r e form. A s the p r o t e i n
F. LeJbvre and others
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(a)
(b) 1
2
1
=
2
"
,
"
(d)
(c) 1
.....
2
1
3
2
200K~
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92.5K~ 69K
46K
30K
20.1K~
-
....
-
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..... 14.4K~
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Fig. 2. Characterization (by SDS P A G E and Western blot analysis) and purification of porcine IFN-ct 1 expressed in E . c o l i C A G 11399 cells harbouring various expression plasmids. (a) Cells bearing plasmid pD1APolFN-ctl were grown at a restrictive temperature (30 °C) in the enriched medium described in Methods to an OD6o o of 1-5. The culture was maintained at the same temperature (lane 1) or at an induction temperature (42 °C, lane 2) for 2 h. Proteins were analysed on a 12.5~ SDS polyacrylamide gel and stained with Coomassie blue. (b) Western blot analysis of the gel in (a) using the C8-18 MAb. Lanes 1 and 2 refer to homologous samples of the gel in (a). The arrow indicates the position of the 28K cr~flgal' IFN-cd fusion protein. Other minor bands probably correspond to degradation products. (c) Cells bearing plasmid pLD67MetlFN-cd were grown and induced as in (a) except that induction was performed at an OD600 of 2. Lane l, non-induced culture; lane 2, induced culture. Cells bearing pLD67MetIFN-cdAHS were grown overnight at 37 °C in the same medium (lane 3). Bacterial proteins were separated on a 15~ SDS-polyacrylamide gel and Western blot analysis was performed as in (a). (d) Purification of mature porcine rlFN-cd. Proteins were analysed on a 15~ SDS polyacrylamide gel and stained with Coomassie blue. Lane 1, total proteins from cells harbouring plasmid pLD67MetlFN-cdAHS; lane 2, approximately 2 gg of immunoaffinity-purified porcine rlFN-~I. Mr standards are indicated on the left of (a), (b), (c) and (d).
subjected to the Edman degradation was not reduced or alkylated, the first N-terminal cysteine residue did not give a P T H derivative. We thus obtained: none-D-L-PQ-T-H-S-L-A in the first 10 steps which clearly corresponds to the terminal sequence deduced from the D N A (Lef~vre & La Bonnardi6re, 1986). On account of the yield of the first step of the Edman degradation (our result was 27~), which was within the normal range of yields (20 to 50~), and as no methionine-PTH was found, we think that at least a major fraction of rIFN-cd was non-methionylated and unblocked at its N terminus; however we cannot exclude the existence of a minor fraction, methionylated and blocked at its N terminus.
In vitro biological properties of rlFN-aI (i) Antiviral effect on different cells The antiviral spectrum of rIFN-~I was compared with that of PoIFN-Le obtained by infection of pig peripheral blood lymphocytes with influenza virus (La Bonnardi6re et al., 1986). For that purpose, we used a panel of five cell lines belonging to four different species (Table l). It appeared that, like IFN-Le, rIFN-al exhibited a broad spectrum of antiviral activity ( 1 0 ~ activity on mouse cells as compared to M D B K cells). But interestingly, rlFN-~I was clearly more active (by sixfold) than its natural counterpart on homologous porcine cells. This
Recombinant porcine IFN-~
Table 1. Antiviral activity of porcine IFN-Le and rlFN-cd on cell lines fi'om different species*
Table 3. Neutralization of porc&e IFN antiviral activity* by an anti-human IFN-~ serum
Antiviral activity (IU/ml) Species
Cell line
IFN-Le
rlFN-~I
Bovine Human Murine Porcine
MDBK WISH L929 PD5 ST83
3000 330 440 330 110
3000 330 250 2200 660
* The antiviral titres of porcine IFN-Le and rlFN-c~l preparations having the same titre on MDBK cells (3000 IU/ml) were determined also on human, murine and porcine cell lines.
Table 2. Antiviral and cytocidal activities of porcine and human IFNs on low-passage porcine kidney cells (RPa 5495)* Cells MDBK IFN PolFN-Le rlFN-cd HulFN-Le
Anti-human IFN-~ serumS" + + +
RPa 5495
AVA
AVA
CCA
8 2 10 4.5 8.5 1
7
