WO1985002862A1

WO1985002862A1 - PRODUCTION OF HUMAN INTERFERON-alpha

Info

Publication number: WO1985002862A1
Application number: PCT/AU1984/000263
Authority: WO
Inventors: Anthony William Linnane; Phillip Nagley; Sangkot Marzuki; Manfred Werner Beilharz; Ian Thomas Nisbet
Original assignee: Monash University; Commonwealth Serum Laboratories Commission
Priority date: 1983-12-23
Filing date: 1984-12-20
Publication date: 1985-07-04
Also published as: EP0165942A1

Abstract

A DNA molecule which on expression codes for a human interferon-alpha, designated IFN-alphaM1, comprises a nucleotide sequence substantially as shown in Figure 2. A recombinant DNA molecule, a cloning vehicle or vector, and a host cell, all containing this nucleotide sequence, are also disclosed. Also disclosed is a polypeptide having human interferon-alpha activity, comprising an amino acid sequence substantially as shown in Figure 2.

Description

PRODUCTJOF OF HϋM IETTERFEROF-α.

This invention relates to a human . interferon-α and to the production thereof. In particular, the invention relates to a complete nucleotide sequence of a human interferon-α gene, and recombinant DNA molecules comprising this nucleotide sequence, as well as processes utilising said recombinant DNA molecules for producing the human interferon-α.

The human interferons are a group of proteins possessing potent antiviral, antiproliferative and immune response modulating activities (1) . In view of the potential therapeutic value of the interferons, together with their limited availability from natural sources, considerable effort has been directed towards the cloning and expression of interferon genes. Three distinct types of interferon, α, β and γ, have been described, based on differences in antigenicity and biological characteristics of the molecules (for review, 2) .

Sequencing and expression studies of interferon-α (IFN-α) genes have been carried out using both cDNAs derived from induced leukocytes and DNA from human chromosomal libraries. The results obtained indicate that the human genome contains at least thirteen functional, non-allelic IFN-α genes as well as a number of allelic variants and pseudogenes (3) . Complete nucleotide sequences for some IFN-α coding regions have been published, eight derived from cDNA clones and seven from clones of genomic DNA (4,5,6,7,8,9, 9a, 9b), and comparison of the nucleotide sequences of different IFN-α coding regions reveals a high degree of homology (88 to 98%) . Differences in the DNA sequence of flanϊing regions and the location of IFN-α genes in tandem array on a single chromosomal fragment have been the basis for suggesting that the genes are non-allelic (3) .

The present invention relates to an IFN-α gene which has been isolated from a human genome library using oligonucleotide probes. The gene, designated IFN-αMl, has been expressed in E. coli using the Ml3 phage vector, and its nucleotide sequence has been ascertained. This data represents the first complete nucleotide sequence published for this IFN-α genetic locus.

According to a first aspect of the present invention, there is provided a DNA molecule which on expression codes for a human interferon-α, comprising a nucleotide sequence substantially as shown in Figure 2.

It will be appreciated that the nucleotide sequence of this aspect of the invention may be obtained from natural, synthetic or semi-synthetic sources; furthermore, the nucleotide sequence may be a naturally-occurring sequence, or may be related by mutation, including single or multiple base substitutions, deletions insertions and inversions, to such a naturally-occurring sequence, provided always that the DNA molecule comprising such a sequence is capable of being expressed as the desired amino acid sequence. The nucleotide sequence may have expression control sequences positioned adjacent to it, such control sequences being derived either from homologous or heterologous sources.

The nucleotide sequence of IFN-αMl according to this invention is further characterised in having a restriction map substantially as shown in Figure lb.

Of the IFN-α nucleotide sequences previously reported (4,5,6,7,8,9) , the IFN-αMl coding region sequence most closely resembles that of IFN-C (4) . IFN-αMl and IFN-C are 98% homologous at the nucleotide sequence level, however at the level of amino acid homology they differ by seven residues.

Weissmann et al. (3) have published a partial amino acid sequence for an IFN-α denoted α4a (153 of 189 amino acids) and a complete amino acid sequence for another IFN-α denoted α4b. The amino acid sequences were derived from unpublished nucleotide sequences of clones isolated from the same human gene library as that used in the present report (12) . IFN-α4a and IFN-α4b are considered to be allelic as they have similar flanking DNA sequences and, on currently published data, have only two amino acid differences (3) .

The amino acid sequence predicted for IFN-άMl is identical to the 153 amino acids of IFN-α4a that have been published (3). Also IFN-αMl differs from IFN-α4b at the same two amino acid residues as IFN-α4a. However, the IFN-αMl coding region contains two restriction enzyme sites (one EcoRII site and one Bspl site_? indicated by asterisks in Fig.lb) which are not present in either IFN-α4a or IFN-α4b (3) . This suggests the existence of the three separate coding regions, IFN-αMl, IFN-α4a and IFN-α4b, in the one individual and hence the presence of at least two genetic loci.

Synthetic oligonucleotides have been used in the screening of cDNA clones (21) but they have not been used extensively in the screening of genome libraries. While the lack of specificity of hybridization presents a problem in the selection of genomic clones with individual oligonucleotides, this can be overcome by using combinations of oligonucleotides. The set of five oligonucleotides used throughout this work (Table 1) was suitable not only for the selection of genomic clones but also for the identification of subclones, for the construction of restriction maps and for priming the chain-termination nucleotide sequencing reactions.

As described in greater detail below, an Alul fragment containing the coding region of the IFN-αMl gene has been inserted into the Hindi site of the phage Ml3 mp 11, resulting in a fusion of the IFN-αMl gene and the β-galactosidase gene. E. coli infected with the recombinant M13 phage carrying the fused gene has been cultured and extracts have shown antiviral activity in cytopathic effect inhibition assays. This antiviral activity was completely neutralised by IFN-α antibodies. In a further aspect of the present invention, there is provided a recombinant DNA molecule which on expression codes for a human interferon-α, comprising a nucleotide sequence substantially as shown in Figure 2, operatively linked to an expression control sequence. The expression control sequence may comprise known initiator and terminator sequences with the interferon nucleotide sequence located between them.

In yet another aspect of this invention, there is provided a recombinant DNA cloning vehicle or vector capable of expressing a human interferon-α, having inserted therein a nucleotide sequence substantially as shown in Figure 2, operatively linked to an expression control sequence. The cloning vehicle or vector may comprise a known bacteriophage or plasmid. This invention further provides a host cell, such as a known E. coli strain, transformed with a recombinant DNA cloning^'vehicle or a recombinant DNA molecule as described above.

As previously described, the amino acid sequence of IFN-αMl expressed by the nucleotide sequence of Figure 2 can be predicted on the basis of the known genetic code. Accordingly, in yet another aspect of this invention, there is provided a polypeptide having human interferon-α activity, comprising an amino acid sequence substantially as shown in Figure 2. This polypeptide may comprise either the pre-IFN-α sequence of 189 amino acids and containing a secretion leader of 23 amino acids, as shown in Figure 2, or the mature IFN-α sequence of 166 amino acids as shown. Finally, this invention provides a method of producing a polypeptide having human interferon-α activity, which comprises the steps of culturing a host cell as described above, and recovering said polypeptide from, the culture.

The invention will be further described by way of reference to the accompanying drawings, in which:

Figure la shows the restriction map of the λMl PstI fragment containing the IFN-αMl gene. Restriction sites are indicated by the symbols τQ, Pstl_?- τ , EcoRI; f_r Hindlll. The hatched area indicates the IFN-αMl coding region. The direction of transcription is from left to right.

Figure lb shows the restriction map of the λMl Rsal fragment containing the IFN-αMl gene and the strategy for sequencing the IFN-αMl gene. Restriction sites are indicated by the symbols:<>, Rsal;<^*, Bspl; ^r, Alul; , Sau3AI; Δ , EcoRII; φ, Hindlll. Arrowed segments below the map indicate the extent and direction of nucleotide sequence data obtained from M13 subclones. The asterisks indicate the Bspl and EcoRII sites which are present in IFN-αMl but absent from both IFN-α4a and IFN-α4b (see below) .

Figure 2 shows the nucleotide and predicted amino acid sequence of IFN-αMl. The initiation codon for pre-interferon, the codon for the N-terminal amino acid of the mature interferon and the termination codon are underlined. The putative 'TATA' box is underlined twice-. Figure 3 shows the nucleotide sequence of the M13 recombinant phage M13-αMl-Bl in the region of the fusion between the β-galactosidase gene of Ml3mpll and the IFN-αMl gene. The numbers' and amino acid sequences refer to segments derived from the β-galactosidase N-terminus, the M13m.pl1 polylinker, the IFN-αMl leader and the N-terminus of the mature IFN-αMl protein.

Materials and Methods

Synthetic oligonucleotides.

Oligonucleotides were synthesized by the solid-phase phosphotriester method (10) and purified by HPLC on a Partisil 10 SAX column operated at ambient temperature and eluted with a gradient of potassium phosphate, pH 6.5, from ImM in 5% acetonitrile to 0.2M in 30% acetonitrile. The nucleotide sequences of these oligonucleotides and the positions at which they are complementary to IFN-α sequences are presented in Table 1. Oligonucleotide probes were 5'-end labelled using T4 polynucleotide kinase (Boehringer-Mannheim) and [γ- ^P]ATP (Amersham) (11) . Unincorporated label was separated from the probes by polyacrylamide gel electrophoresis.

Screening of human genome library.

A human- genome library in phage γ Charon 4A prepared by Lawn and colleagues (12) was used. Approximately 300,000 plaques were screened by the 'amplified plaque lift* procedure of Woo (13) . The hybridization temperatures used are indicated in Table 1. Analysis, of subclones.

Restriction fragments of the γ clones were inserted into plasmid pϋC9 (14) and cloned in E. coli

— + + — ED8654 [SupE, SupF, hsdR m S ,-met , trpR] . Colonies were screened by the Grunstein-Hogness colony hybridization method (15) . Subsequently, restriction fragments of the selected pϋC9 recombinant were inserted into Ml3mp9 or M13mpll (16) and used to transform E. coli JMlOl [ lacpro, thi, supE, F'traD36, proAB, LacI^Z Ml5] . Ml3 recombinant plaques were screened by the Benton and Davis procedure (17) .

Hybridization was carried out using the synthetic oligonucleotide probes (Table 1) . Restriction maps were constructed using standard methods, including-

Southern blotting (18) .

DNA sequencing.

Single-stranded DNA was prepared from M13 subclones and sequenced by the dideoxy chain-termination method of Sanger et al. (19) . Priming was carried out using synthesized oligonucleotides, either the Ml3 'universal primer* (5'-GTAAAACGACGGCCAGT-3*) or an IFN-α gene-specific oligonucleotide (Table 1) .

Expression of cloned IFN-α DNA.

The conditions for infection,of E. coli with the recombinant phage and inductio with isopropyl β-D-thiogalactopyranoside (IPTG) were as previously reported (20) , except that JMlOl was used as the host strain.

Interferon assays. A standard cytopathic effect (CPE) inhibition assay using human HEp-2 cells and Semliki forest virus was used (for review, 1) .

RESULTS

A human genome library in phage λ Charon 4A (12) was screened for the presence of IFN-α genes with synthetic oligonucleotides. The sequences of the oligonucleotide probes correspond to a number of different, highly conserved segments within published IFN-α coding regions (Table 1) . Using the individual probes approximately 300,000 recombinant phage were screened, resulting in the isolation of 297 putative IFN-α clones. The number of putative positive clones was reduced to twenty-eight by using combinations of the oligonucleotide probes. One clone, designated _λMl, which hybridized to all five oligonucleotide probes (Table 1) , was selected for detailed analysis. A PstI fragment of the-_λMl DNA to which the oligonucleotide probes hybridized was subcloned into pϋC9 by standard methods. Following amplification in E. coli the purified PstI fragment was digested with selected restriction enzymes and the resulting fragments were separated by electrophoresis on an agarose gel. The fragments were transferred to nitrocellulose paper, hybridized to specific oligonucleotide probes and a restriction map of the PstI fragment was derived (Fig.la) .

Digestion of the PstI fragment with Sau3AI resulted in four fragments: a 790 base pair (bp) fragment which hybridized to oligonucleotide probe 5, a 176 bp fragment which hybridized to probe 4, a 269 bp fragment which hybridized to probes 2 and 3, and a 74 bp fragment which hybridized to probe 1. All four fragments were cloned in both orientations into the BamHI site of the vector M13mp9 (Fig.lb). An Rsal fragment (977 bp) , wholly contained within the PstI fragment and hybridizing to all five probes, was similarly cloned in both orientations into the Hindi site of the vector M13mpll (Fig.lb) . The identification of recombinant clones of interest and the determination of the orientation of the inserted fragments was achieved by screening the M13 recombinant clones with the appropriate oligonucleotides. Utilizing either the M13 'universal primer' or an IFN-α-specific oligonucleotide as the primer, the nucleotide sequence of these M13 recombinant subclones was obtained by the dideoxy chain-termination method. The sequence determined is shown in Fig.2 and a detailed restriction map derived both from this sequence and restriction enzyme analysis is shown in Fig.lb. Comparison with previously reported IFN-α sequences reveals that the nucleotide sequence of the Rsal fragment contains an entire IFN-α coding region. This coding region specifies a pre-IFN-α of 189 amino acids, consisting of a 23 amino acid leader and a 166 amino acid mature IFN-α protein (Fig.2) .

In order to demonstrate that the IFN-αMl gene codes for a biologically active, product, the gene was expressed in E. coli. An Alul-fragment of 669 bp (Fig.lb) was cloned into the Hindi site of Ml3mpll and clones with the correct orientation of the Alul fragment were selected by hybridization with oligonucleotides 4 and 5 (Table 1) . One such clone, M13-αMl-Bl, was subjected to sequence analysis and shown to have the predicted fused gene. Ml3-αMl-Bl contained the β-galactosidase promoter and the N-terminal 15 nuσleotides of the β-galactosidase gene coding region, 20 nucleotides of the M13mpll polylinker sequence, and 25 nuσleotides of the IFN-αMl leader sequence followed by the mature IFN-αMl coding region (Fig.3) . In the fused protein product, it would therefore be predicted that the 23 amino acid interferon leader would be replaced by a 19 amino acid leader (11 residues of which are non-interferon) , assuming the N-terminal methionine is removed from the β-galactosidase N-terminus.

Cultures of E. coli (JMlOl) were infected with the recombinant phage M13-αMl-Bl and induced with the lac operon inducer IPTG. CPE inhibiton assays for ς antiviral activity detected 6.3 x 10 IU/1 of culture in the spent culture supernatant. Extracts of the pelleted cells contained 1.3 x 10 IU/1 of culture.

The interferon activity in both the culture supernatant and in the cell pellet extracts' was completely neutralized by both a polyclonal antiserum against human IFN-α (Cantell) and a monoclonal anti-human IFN-α antibody. It may be noted that the level of interferon expression obtained was lower than that previously reported with a M13 vector (20) .

Factors which may account for this difference include the intrinsic specific activities of the particular interferons, the specific activity of the product of the particular fused gene constructed here

(Ml3-αMl-Bl) , and the properties of the host E. coli strain. Table 1. Synthetic oligonucleotides used in the screening of the human genome library and the characterization of IFN-α genes

"V

(a) Nucleotide posi tion 1 of the IFN gene is taken as being the 'A' of the ΛTG coding for translation ini tiation in the IFN-αl gene (9) .

(b) The temperature gi ven indicates the empirical ly determined temperature of hybridi zation and washing for the ol igonucleotide probes .

REFERENCES

1. Stewart II, W. (1979) The Interferon System; Springer, New York.

2. Lengyel, P. (1982) Ann. Rev. Biochem. 51, 251-282.

3. Weissmann, C, Nagata, S., Boll, W. , Fountoulakis, M. , Fujisawa, A., Fujisawa, J., Haynes, J., Henco, ., Mantel, N., Ragg, H., Scheiπ, C, Schmid, J., Shaw, G._r Streuli, M., Taira, H. , Todokoro, K. and Weidle,

-(1982) Interferons, UCLA Symposia on Molecular and Cellular Biology, Vol.XXV; T. Merrigan and R. Friedman, Eds., pp.295-326, Academic Press, New York.

4. Goeddel, D., Leung, D., Dull, T., Gross, M. , Lawn, R., McCandliss, R., Seeburg, P.-, Ullrich, A., Yelverton, E. and Gray, P. (1981) Nature 290, 20-26.

5. Lawn, R., Adelman, J., Dull, T., Gross, M., Goeddel, D. and Ullrich, A. (1981) Science 212, 1159-1162.

6. Lawn, R., Gross, M., Houck, C., Franke, A., Gray, P. and Goeddel, D. (1981) Proc. Natl. Acad. Sci. USA 78, 5435-5439.

7. Streuli, M., Nagata, S. and Weissmann, C. (1980) Science 209, 1343-1347

8. Mantei, N.,- Schwarzstein, M., Streuli, M., Panem, S., Nagata, S. and Weissmann, C. (1980) Gene 10, 1-10.

9. Nagata, S., Mantei, N. and Weissmann, C. (1980) Nature 287,- 401-408.

10. Duckworth, M., Gait, M., Goelet, P., Hong, G., Singh, M. and Titmas, R. (1981) Nucleic Acids Res. 9, 1691-1706.,

11. Wallace, R., Shaffer, J., Murphy, R., Banner, J., Hirose, T. and Hakura K. (1979) Nucleic Acids Res. 6, 3543-3557.

12. Lawn, R., Fritsch, E., Parker, R., Blake, G. and Maniatis, T. (1978) Cel 15, 1157-1174.

13. Woo, S. (1980) Methods in Enzy ology 6S, 339-395.

14. Vieira, J. and Messing, J. (1982) Gene 19, 259-268.

15. Grunstein, M. and Hogπess, D. (1975) Proc. Natl. Acad. Sci. USA 72, 3961 3965.

16. Messing, J. and Vieira, J. (1982) Gene 19, 269-276.

17. Benton, W. and Davis, R. (1977) Science 196, 180-182.

18. Southern, E. (1975) J. Mol . Biol . 98, 503-517.

19. Sanger, F., Nickleπ, S. and Coulson, A. (1977) Proc. Natl. Acad. Sci. US 74, 5463-5467.

20. Slocombe, P., Eastoπ, A., Boseley, P. and Burke, D. (1982) Proc. Natl. Acad. Sci. USA 79, 5455-5459.

21. Goeddel, D., Yelverton, E., Ullrich, A., Heyncker, H., Miozzari, G., Holmes, W., Seeburg, P., Dull, T., May, L., Stebbing, N., Crea, R., Maed S., McCandliss, R., Sloma, A., Tabor, J., Gross, M., Familletti, P. and Pestka, S. (1980) Nature 287, 411-416. It will be appreciated that many modifications and variations may be made to the particular methods described above by way of illustration of the present invention, and that the present invention includes all such modifications which fall within the scope of the invention as broadly described above.

Claims

CLAIMS :

1. A DNA molecule which an expression codes for a human interferon-α, designated IFN-αMl, comprising a nucleotide sequence substantially as shown in Figure 2.

2. A DNA molecule according to claim 1, wherein the nucleotide sequence isfurther characterised in having a restriction map substantiallly as shown in Figure lb.

3. A recombinant DNA molecule which on expression codes for a human interferon-α, designated IFN-αMl, comprising a nucleotide sequence substantially as shown in Figure 2, operatively linked to an expression control sequence.

4. A recombinant DNA molecule according to claim 3, wherein the expression control sequence comprises initiator and terminator sequences with said nucleotide sequence located between said initiator and terminator sequences.

5. A recombinant DNA cloning vehicle or vector capable of expressing a human interferon-α, having inserted therein a nucleotide sequence substantially as shown in Figure 2, operatively linked to an expression control sequence.

6. A cloning vehicle or vector according to claim 5, wherein the vehicle or vector is a bacteriophage.

7. A host cell, transformed with a recombinant DNA cloning vehicle or vector according to claim 5, or a recombinant DNA molecule according to claim 3.

8. A host cell according to claim 7, wherein said host cell is a known strain of E.coli.

9. A polypeptide having human interferon-α activity, comprising an amino acid sequence substantially as shown in Figure 2.

10. A polypeptide according to claim 9, comprising a pre-IFN-α sequence of 189 amino acids, including a secretion leader of 23 amino acids, substantially as shown in Figure 2.

11. A polypeptide according to claim 9, comprising a mature IFN-α sequence of 166 amino acids, substantially as shown in Figure 2.

12. A method of producing a polypeptide having human interferon-α activity which comprises the steps of culturing a host cellaccording to claim 7, and recovering said polypeptide from the culture.

13. A polypeptide having human interferon-α activity, produced by the method according to claim 12.