NO851065L - GENETIC TECHNOLOGY PROCEDURE FOR MANUFACTURING HUMAN GAMMA INTERFERON AND IMPLEMENTATION FOR IMPLEMENTING THIS PROCEDURE - Google Patents

Description

The invention relates to a method for production

of human-gamma interferon, chemically synthesized genes,

which encodes this peptide, as well as suitable vector constructs and host organisms for production of this polypeptide.

The gamma interferon (formerly immune interferon or type II interferon; here briefly referred to as IFN-γ) was discovered

in the year 1965 of; F. Wheelock (Wheelock; Science 149 (1965), 310), who showed that IFN-γ can protect certain cells against a viral infection. Human IFN-γ (for background see W.E.Stewart, II, The INterferon System, Springer Verlag

(2nd ed. 1981)), is a polypeptide of 146 amino acids (Gray

et al., Nature 295 (1982), 503), which are naturally glycosylated. The glycoprotein has a molecular weight of approx. 63,000 - 73,000 (Pestka et al., J. Biol. Chem 258 (1983), 9706) and exists in its functional form probably as a tetramer. The glycosylation of IFN-γ is not required for its functionality. Thus, the glycosidase treatment of IFN- does not reduce its anti-viral activity in human fibroblast cell cultures (Keller et al., J. Biol. Chem. 258 (1983), 8010).

Furthermore, unlike alpha-interferons and beta-interferon, IFN-y is unstable at pH 2 and is also inactivated by heat (60°C).

The development of human IFN-γ from cell cultures of human cell lines or from leukocytes (blood preserves) is only possible with poor yield and low product purity. Genetic engineering methods for the production of IFN-y-like polypeptides have therefore already been proposed, for example in European patent application with publication no. 95,350 the chemical synthesis of a gene that codes for IFN-γ, its incorporation into a hybrid plasmid, the transformation of E.coli and the production of an immunologically active polypeptide.

Human IFN-Y has the following peptide sequence (Devos et al., Nucl. Acids Research 10 (1972) 2487):

The present invention relates to the genetic engineering production of human IFN-Y by means of a chemically synthesized gene, which is characterized by vec. The DNA sequence I (Appendix). • The genetic code is known to be "degenerate", i.e. a single nucleotide sequence is coded for only two amino acids, while the remaining 18 genetically codeable amino acids are to be assigned 2 to 6 triplets. Furthermore, the host cells from different species do not always make the same use of the variation possibilities provided by this. For the synthesis of the genes, there is thus an unmanageable diversity of coding possibilities. It was now found that the DNA sequence I (appendix) codes for

the total amino acid sequence 1-14 6, both scrn and the DNA partial sequences used for the synthesis of sequence I,

is particularly advantageous for the genetic engineering synthesis of IFN-Y- At the 5'-end of the coding strand of DNA sequence I, an "overhanging" DNA sequence, for example corresponding restriction endonuclease Eco RI, is joined at the 3<1->end of the coding string, on the other hand, a single string,

overhanging sequence which is advantageously different from that of the 5' end, for example corresponding to the restriction enzyme Sal I. These two different recognition sequences guarantee the introduction of DNA into plasmids in the desired orientation. Equally overlapping sequences can also be selected and a corresponding selection made later.

Between these recognition sequences and the codes for the amino acid sequence, the amino acid methionine is located at the 5<1> end of the coding strand in the code. Alternatively, there can be a presequence (also called single or "leader" sequence) of a bacterial or otherwise host-specific protein (review article: Perlman and Halvorsen; J. Mol. Biol. 167 (1983), 391), which for example causes secretion of the desired polypeptide from the cytoplasm and in this excretion process is cleaved from a naturally occurring signal peptidase in the host cell. In addition to triplet 146, which codes for glutamine, then follows a stop codon, or preferably two stop triplets.

Two internal, singular cut sites for the restriction enzymes Barn HI and Hind III (in codon 31 and 94 respectively

in the coding strand or in codon 32 or 95 respectively

in the non-coding strand), enables the subcloning of three gene fragments IFN-I, IFN-II and IFN-III (see DNA sequence II), which can be incorporated into well-studied cloning vectors, such as e.g. pBR 322 or pUC 8. In addition, a number of other, unique recognition sequences were built into the structural gene as restriction enzymes, which on the one hand provide access to partial sequences of IFN- and on the other hand allow the implementation of variations:

The gene, which consists of DNA sequence I, the codon for methionine

and the above ends can be built up from 34 oligonucleotides with a length of 18 to 33 nucleotides (see DNA sequence II), as these are first chemically synthesized and then joined enzymatically over "sticky ends" of 4-6 nucleotides.

In the case of DNA sequence I, consideration was also given to the fact that

the amino acids that can be assigned to several codons are not equivalent, but rather show different preferences in the relevant host cell, such as E.coli. Furthermore, palindomic sequences were reduced to a minimum measure.

The gene structure of DNA sequence I is thus easily accessible from relatively small building blocks, enables the subcloning of 3 gene fragments in well-known vectors and enables their combination into the total gene as well as any changes thereof.

The incorporation of the synthetic genes or gene fragments into cloning vectors, for example in the commercially available plasmids pUC 8 and pBR 322, or other commonly available plasmids such as ptac 11 and pKK 177.3, takes place in a manner known per se. The chemically synthesized genes can also be equipped in advance with suitable chemically synthesized control regions, which enable the production of the proteins. Reference should be made in this regard to the textbook by Maniatis (Molecular CLoning, Maniatis et al., Cold Spring Harbor, 1982). The transformation of the hybrid plasmids thus obtained in suitable host organisms, advantageously E. coli, is likewise known in and of itself, and is described in detail in the aforementioned textbook. The recovery of the produced protein and its purification are also described (J.A. Georgiades, Texas Reports in Biology and Medicine et al (1981) 179; Came and Carter (eds), "Interferons and Their Applications", Springer-Verlag 1984).

The gene fragments IFN-I, IFN-II and IFN-III, which can be obtained according to the invention, the thus obtained hybrid plasmids and the transformed host organisms are new and subject of the invention. The same applies to the new DNA sequences which have been converted from DNA sequence I. Other embodiments of the invention are specified in the patent claims.

In the following examples, some embodiments of the invention are explained, from which a number of possible transformations and combinations are apparent to the person skilled in the art. Percentages are hereby calculated on the basis of weight, unless otherwise stated.

Examples.

1. Chemical synthesis of a single-stranded oligonucleotide.

Using the example of gene building block Ia, which comprises nucleotides 1-23 in the coding strand, the synthesis of the gene building blocks is explained. According to known methods (M.J.Gait et al., Nucleic Acids Res. 8 (1980) 1081-1096)), the nucleoside at the 3'-end, in the present case i.e. cytidine (nucleotide no. 23), is covalently bound over 3 The '-hydroxy function on silica gel ((R)Fractosil, company Merck). In order to achieve this, the silica gel is first reacted with 3-(triethoxysilyl)-propylamine, whereby a Si-O-Si bond is formed, while ethanol is removed. The cytidine is reacted as N-4benzoyl-31 - 0-succinoyl-5<1->dimethoxytriethyl ether in the presence of paranitrophenol and N,N<1->dicyclohexylcarbodiimide with the modified carrier, whereby the free carboxy group in the succinoyl group acylates the amino residue in the propylamino group.

In the following synthesis steps, the basic components such as 5<1->O-dimethoxytrityl-nucloside-3<1->phosphoric acid monomethyl ester, dialkylamide or -chloride are introduced, whereby the adenine is present as N 6 compounds, the cytosine as an N 4 compound, the guanine as N 2 compound and the thymine containing no amino group, without protecting group.

50 mg of the polymeric carrier, which bound contains 2

(imol cytosine, treated in sequence with the following reagents:

a) Nitromethane,

b) saturated zinc bromide solution in nitromethane with 1% water,

c) methanol,

d) tetrahydrofuran,

e) acetonitrile,

f) 40 µmol of the corresponding nucleoside phosphate and 200 µmol tetrazole in 0.5 ml anhydrous acetonitrile (5 min.), g) 20% acetic anhydride in tetrahydrofuran with 40% lutidine 10% dimethylaminopyridine (2 min.),

h) tetrahydrofuran,

i) tetrahydrofuran with 20% water and 40% lutidine,

j) 3% iodine in collidine/water/tetrahydrofuran in the volume ratio 5:4:1,

k) tetrahydrofuran and

1) methanol.

By "phosphite" here is meant deoxyribose-3<1->monophosphoric acid monomethyl ester, whereby the third valence is saturated with chlorine or a tertiary amino group, for example a morpholino residue. The yields of the individual synthesis steps can optionally be determined after the detritylation reaction b) spectrophotometrically by measuring the absorption of the dimethoxytrityl cation at a wavelength of 496 nm.

After the synthesis of the oligonucleotide is completed, the methyl phosphate protecting groups in the oligomer are cleaved off with the help of p-thiocresol and triethylamine.

The oligonucleotide is then separated from the solid support by a 3-hour treatment with ammonia. A 2- to 3-day treatment of the oligomers with concentrated ammonia quantitatively cleaves the amino protecting groups from the bases.

The crude product thus obtained is purified by high-pressure liquid chromatography (or by polyacrylamide gel electrophoresis. The other gel building blocks Ib-IIIl are also synthesized in a completely similar way, the nucleotide order of which appears from the DNA sequence

II.

2. Enzymatic joining of the single-stranded oligonucleotides to the gene fragments IFN-I, IFN-II and IFN-III.

For phosphorylation of the oligonucleotide at the 5' end,

1 nmol of each of oligonucleotides Ia and Ib treated with 5 nmol adenosine triphosphate with four units of T4-polynucleotide kinase in 20^1 50 mM Tris-HCl buffer (pH 7.6), 10 mM magnesium chloride and 10 mM dithiothreitol ( DTT) for 30 minutes at 37°C (C.C.Richardson, Progress in Nucl. Acids Res. 2

(1972) 825). The enzyme is inactivated by heating to 95°C for 5 minutes. Oligonucleotides Ia and Ib are then hybridized against each other, heating in aqueous solution for 2 minutes to 95°C and then slowly cooling to 5°C.

Analogously, the oligonucleotides Ic and id, le and

If, lg and Ih as well as li and Ij, and are hybridized in pairs. For the IFN-II subfragment, the oligonucleotides are phosphorylated

Ila with IIb etc., to Ilk with III and for subfragment IFN-III the oligomers Illa with Illb etc. to Ulk with IIIl and hybridized in pairs.

The thus obtained five oligonucleotide pairs for the gene fragment IFN-I respectively the six oligonucleotide pairs for the gene fragments IFN-II and IFN-III are all ligated as follows: The double-stranded nucleotides are combined and ligated in each 40 (in 50 mM Tris-HCl buffer, 20 mM magnesium chloride, and

10 mM DTT with the help of 100 units of T4 DNA lgase at 15°C

within 16 hours. The purification of the gene fragments IFN-

I to IFN-III takes place by gel electrophoresis on a 10% polyacrylamide gel (without urea addition, 20.40 cm, 1 mm thickness), whereby ØX 174 DNA (Fa. BRL), cut with Hinf I, or pBR 322 served as marker substance , the cut with Hae III. 3. Production of hybrid plasmids, which contain the gene fragments IFN-I, IFN-II and IFN-III.

a) Incorporation of the gene fragment IFN-I into pBR 322.

The commercially available plasmid pBR 322 is opened in a known manner

with the restriction endonucleases Eco RI and Ban HI as specified by the manufacturer. The digestion batch is divided on a 5% polyacrylamide gel by electrophoresis in a known manner and the fragments are made visible by staining with ethidium bromide or by radioactive labeling ("Nick-Translation" according to Maniatis, see above). The plasmid bands are then removed from the acrylamide gel and electrophoretically separated from polyacrylamide. The division of digestive

the batch can also take place on 2% low-melting agarose gels (as described in example 5a).

i ug plasmid is then ligated with 10 ng gene fragment (IFN-I overnight at 16°C. The hybrid plasmid according to fig.

1 is achieved.

b) Incorporation of the gene fragment IFN-II into pUC 8 analogously to a) the commercially available plasmid pUC 8 is divided with

Barn HI and Hind III and ligate with the bone fragment IFN-II. The hybrid plasmid according to fig. 2 is achieved.

c) Incorporation of the gene fragment IFN-II into pUC 8 analogous to a) dividing plasmid pUC 8 with Hind III and Sal

I, and ligated with gene fragment IFN-III. The hybrid plasmid according to fig. 3 is achieved. 4. Synthesis of the complete gene and incorporation into a plasmid.

a) Transformation and enlargement.

The thus obtained hybrid plasmids are transformed in E.coli.

To achieve this, the strain E. coli K 12 is made competent by treatment with a 70 mM calcium chloride solution and the suspension of the hybrid plasmid is added in 10

mM Tris-HCl buffer (pH 7.5), which is 70 mM of calcium chloride. The transformed strains are selected as usual using the antibiotic resistances, respectively. - sensitivities that are mediated by the plasmid and the hybrid vectors are magnified. After killing the cells, the hybrid plasmids are isolated, split with the originally used restriction enzymes and the gene fragments IFN-I, IFN-II and IFN-III are isolated by gel electrophoresis.

b) Joining of the gene fragments.

The subfragments IFN-I, IFN-II and IFN-III obtained

during the enlargement, are linked together enzymatically as described in example 2, and the thus obtained synthetic

gene with DNA sequence I is introduced into the cloning vector pUC 8. A hybrid plasmid is obtained according to fig. 4. 5. Construction of hybrid plasmids for expression of DNA sequence I.

a) Incorporation in pKK 177.3.

The expression plasmid pKK 177.3 (plasmid ptac 11, Amman

et al., Gene 25 (1983) 167), whereby a sequence containing a Sal-I cut site is synthetically incorporated into the Eco RI recognition site), is opened with the restriction enzymes Eco RI and Sal I. From the plasmid according to fig. 4, introduction I is excised with the restriction enzymes Eco RI and Sal I.

Insert I is added to 2%, low-melting agarose, separated from plasmid DNA and the insert is recovered by dissolving the gel at an elevated temperature (as specified by the manufacturer). By ligation of the cleaved plasmid pKK 177.3 with Gel I, a hybrid plasmid is obtained, in which the introduction is sealed in front of an expression or regulatory area.

After the addition of a suitable inducer such as Isopropyl-B-thiogalactopyranoside (IPTG), an mRNA is formed, which leads to the production of the methionyl polypeptide corresponding to DNA sequence I.

b) Integration in pMX 2.

The expression plasmid pMX2 consists of a pUC-8 plasmid

which is shortened by 21 nucleotides and is prepared in the following way: pUC 8 is opened with restriction endonuclease Eco RI and then treated with exonuclease Bal 31 under conditions which allow cleavage of approx. 20 nucleotides on both sides of the Eco RI cut site (Maniatis, see

above). Subsequently, any remaining ends of the thus treated plasmid are filled in with Klenow DNA polymerase, the plasmid is post-cut with restriction endonuclease Hind III and the plasmid is purified over 1% low-melting agarose gels according to instructions from the manufacturer. In the plasmid, the polylinker that was originally present in pUC 8 and that was restricted by restriction enzyme cutting Eco RI and Hind III is reintroduced,

and which was destroyed during the manipulations described above. To achieve this, pUC 8 is opened with the restriction enzyme Eco RI and the excess ends are filled in with Klenow DNA polymerase using 32p-labeled nucleoside triphosphates. The polylinker is then excised from the plasmid with the restriction enzyme Hind III and separated from the plasmid by electrophoresis on 10% acrylamide gels. After identification of the polyfor linker band by means of an autoradiography, the poly linker is freed from acrylamide residues by electroelution and ligated into the shortened pUC 8 plasmid. The thus constructed plasmid pMX 2 is then opened with the restriction enzymes Eco RI and Sal I and ligated into the expression plasmid pMX 2 with Y-interferon gel I, which exhibits Eco RI and Sal I recognition sequences at the ends (Fig. 5). Clones exhibiting a high interferon titer are then identified on the basis of the biological activity determination.

6. Transformation of hybrid plasmids.

Competent E.coli cells are transformed with 0.1 to 1 µg

of hybrid plasmids, containing sequence I, and plated on agar plates containing ampicillin. Subsequently, clones containing the correctly integrated γ-interferon gene sequence in the corresponding plasmids can be identified by rapid DNA processing (Maniatis,

see above).

7. Preparation of polypeptides exhibiting β-interferon activity.

After transformation of the aforementioned hybrid plasmids into E.coli, a polypeptide emerges which, in addition to the γ-interferon amino acid sequence, also carries a further methionyl group at the amino end.

8. Processing and cleaning.

The bacterial strains that have been grown to the desired optical density are incubated with a suitable inducer, for example IPTG, for a sufficient time, for example for 2 hours. The cells are then killed with 0.1% cresol and 0.1 mM benzylsulphonyl fluoride. After centrifugation or filtration, the cell mass is taken up in a buffer solution (50 mM Tris,

50 mM EDTA, pH 7.5) and digested mechanically, for example

(R)

with a French press, or ( R ) Dyno-Muhle (Fa. Willy Bachofor, Basel), after which the insoluble components are centrifuged off. From the supernatant liquid, the protein containing the γ-interferon activity is purified using conventional methods. Suitable are ion exchange, adsorption, gel filtration columns or affinity chromatography on anti-body columns. By sodium dodecyl sulfate acrylamide gel or HPLC analysis, the enrichment and purity of the product is checked.

For biological characterization of the product on -interferon activity, indicator cell lines, such as e.g. Vero cells, and incubated with dilution series of interferon-containing bacterial extracts. Then tested with infection with a virus that

VSV (Vesicular Stomatitis Virus), to which dilution level with the Vero cells an anti-viral status can be achieved with the bacterial extract. The exercise can take place microscopically or by determining neutral-red uptake.

y y-interferon activity can also be measured with a commercially available radioimmunoassay (Celltech Ltd.), which is based on a monoclonal antibody against y-interferon.

Claims (3)

1. Genetic engineering method for the production of human gamma interferon, characterized in that a synthetic gene is used, which contains DNA sequence I. 2. Method according to claim 1, characterized in that two stop codons follow the gene. 3. Method according to claims 1 or 2, characterized in that E.coli serves as host organism.