GB2069504A

GB2069504A - Microbiologically Prepared Polypeptide Comprising the Amino Acid Sequence of Human Interferon, DNA and Plasmids which Code for this Sequence, Microorganisms which Contain this Genetic Information, and Processes for their Preparation

Info

Publication number: GB2069504A
Application number: GB8104763A
Authority: GB
Original assignee: Hoechst AG
Current assignee: Hoechst AG
Priority date: 1980-02-16
Filing date: 1981-02-16
Publication date: 1981-08-26
Also published as: ES499289A0; JPS56131522A; FR2482132A1; NL8100719A; BE887530A; EP0034306A2; ES8205429A1; EP0034306B1; ATE26999T1; ZA81973B; AU6728781A; DE3005843A1; DE3176161D1; ES8205430A1; ES8302089A1; IT1141976B; SE8101004L; ES499285A0; ES499290A0; EP0034306A3

Abstract

There is utilised recombinant DNA technology in the production of interferon, which provides a microbiologically produced polypeptide, all or part of which comprises the amino acid sequence of human interferon, especially fibroblast (???) interferon. There is also provided CDNA which codes for such a polypeptide; a plasmid, especially plasmid pBR 322, which comprises the cDNA; and a microorganism, for example Escherichia coli, especially E. coli K12 or E. coli X1776, which comprises the genetic information required for the production of such a polypeptide. The cDNA is preferably prepared using mRNA obtained from human fibroblasts.

Description

SPECIFICATION Microbiologically Prepared Polypeptide Comprising the Amino Acid Sequence of Human Interferon, DNA and Plasmids which Code for this Sequence, Microorganisms which Contain this Genetic Information, and Processes for Their Preparation It has been known for more than 20 years that interferon has antiviral activity. Investigations in recent years have shown, inter alia, that interferon also inhibits cell division and regulates many reactions of the immunological system. All these results underline the great medical importance of interferon. The limited amounts of interferon which it has hitherto been possible to use for scientific and therapeutic investigations have been obtained primarily from fibroblast and lymphocyte cultures. However, it was not possible with either process to prepare relatively large amounts of interferon or even sufficient amounts for answering individual questions. This is the reason why the state of knowledge in the field of interferon is progressing very slowly and why very few of the urgently desired investigations in the field of cancer can be carried out. It is therefore imperative to adopt a new approach to the production of interferon. The present invention relates to an approach utilising recombinant DNA technology. The present invention accordingly provides a microbiologically prepared polypeptide, all or part of which comprises the amino acid sequence of human interferon, especially human fibroblast interferon ({3-interferon). The invention also provides cDNA which codes for a polypeptide as defined above, whenever prepared using mRNA obtained from human fibroblast cells. The invention further provides a plasmid, especially plasmid pBR 322, which comprises cDNA of the invention, and also provides a microorganism, preferably E. coli, and

comprises a plasmid of the invention. The plasmid may be introduced into the microorganism by transformation. The invention provides moreover a process for the production of a polypeptide, all or part of which comprises the amino acid sequence of human interferon, especially human fibroblast interferon, which comprises obtaining the genetic information for the biosynthesis of the polypeptide using fibroblast mRNA, incorporating the resulting gene into microorganisms in a manner which is in itself known, and selecting and culturing, in a manner which is in itself known, those microorganisms which produce the polypeptide. (The term "known" is used herein to denote in actual use in the art or described in the literature of the art.). The process of the invention generally comprises obtaining from human fibroblasts mRNA for the polypeptide, producing double stranded cDNA therefrom, incorporating the cDNA into a plasmid, introducing the plasmid into microorganisms, and selecting and culturing those microorganisms that produce the polypeptide. The invention also provides a process for the preparation of RNA which comprises arranging a caesium chloride-free cell lysate as a layer on an aqueous caesium chloride solution, centrifuging the resulting system, and obtaining the RNA. The lysate is, for example, a lysate of human fibroblasts. The interferon protein formed in the process of the invention possesses the biological and immunological properties which are characteristic of this protein. The following procedure can be adopted to achieve the aim according to the invention, the means illustrated being merely an example of methods which can be used. Human fibroblasts are activated for the maximum production of interferon mRNA, in the presence of a protein synthesis inhibitor, with the aid of polyinosinic acid/polycytidylic acid. The cells are then denaturated and lysed, and the RNA is obtained therefrom as a precipitate after caesium chloride centrifugation. It has been found advantageous to prepare a cell lysate, to arrange this lysate as a layer on an aqueous caesium chloride solution, and to centrifuge the resulting system. in contrast to methods proposed previously, however, it has now been found that the lysate should be caesium chloride-free to achieve the most complete possible separation of the mRNA from the mixed RNAs in the next reaction step. After further precipitation, the mRNA is separated from the resulting mixed RNAs using affinity chromatography, especially an oligo-dtcellulose column. The mRNA obtained is completed to give an RNA-DNA double strand with the aid of the enzyme reverse transcriptase. After digestion of the RNA strand, the DNA strand (complementary DNA=cDNA) is completed to give a DNA-DNA double strand (dsDNA) again using reverse transcriptase, or using the enzyme polymerase I. This double-stranded DNA must now be incorporated into a plasmid. For this purpose, it is necessary to lengthen the 3' end of the DNA, for example with dCMP residues. A plasmid, for example pBR 322 or pBR 325, is cut with a restriction endonuclease, for example, Pstl, and lengthened with, for example, dGMP residues. When the DNA lengthened in this way is brought together with the correspondingly lengthened plasmid, base pairing takes place between the DNA and the plasmid. This circularized molecule is then introduced by transformation into microorganisms, in particular into bacteria, preferably E. coli, for example, E. coli K 12 or E. coli X 1776; the bonds which are not yet closed are covalently linked by means of the enzymes of the microorganism. The transformed microorganisms are plated on agar and selected on the basis of antibiotic resistances which are determined by the chosen plasmid and the restriction endo-nuclease used. Those clones which express interferoncontaining proteins are selected with the aid of immunological and biological tests. These clones are bred and the microorganism mass is centrifuged off, extracted and tested for interferon content. The solution obtained on digestion of the interferon-producing clones contains interferonprotein. The following Example illustrates the invention. 1. Process for the Isolation of RNA a) Preparation of Interferon-producing Fibroblast Cells Human neonatal preputial fibroblasts are reproduced, at 37[deg]C, using a suitable culture medium, for example Eagle's Medium MEM (minimum essential medium) (H. Eagle, Science 130, 432, (1959)) plus foetal calf serum, in narrow-necked cylindrical bottles, (0: 10 cm, length: 40 cm), with several transfers. After developed cell cultures of the 25th to 35th cell passage have been obtained, the reproduction medium is replaced by the interferon-inducing medium. The latter consists of Eagle's MEM, to which a suitable amount of the fibroblast interferon inducer polyinosinic acid/polycytidylic

Microg/ml) and of the protein synthesis inhibitor

case 50 Microg/ml are added.After incubation of the cell cultures at 37[deg]C for 4 to 6 hours (in the present case 5 hours), the culture supernatant is poured off. b) Preparation of RNA The cells obtained are taken up in a "denaturing medium" (4 M in respect to guanidinium thiocyanate, 1 M in respect of mercaptoethanol and 0.15 M in respect of sodium acetate-pH 5.5) and homogenized for 1 minute at 0[deg]C using an Ultra-Turrax. The homogenized mixture is centrifuged at 20,000 rpm (40,000 G) for 15 minutes at 4[deg]C. The supernatant is then arranged in a layer on 5 ml of a solution ("cushion") which is 5.7 M in respect of CsCI, 10 mM in respect of trishydroxyaminomethane (tris) and 10 mM in respect of ethylenediaminetetraacetic acid (EDTA), and which has a pH of 7.6, and is centrifuged in a Beckman-SW 27 Rotor for 36 hours at 20[deg]C and 22,000 rpm. It was found that, in contrast to data in the literature, the addition of CsCI to the lysate must be prevented in order to achieve the most complete separation possible of the mRNA in the following reaction step. After centrifugation has ended, the small Polyallomer tubes are frozen in liquid nitrogen and the bottom of the small tube, where the RNA precipitate is located, is cut off. The precipitate is taken upon in a solution which is 10 mM in respect of tris and 10 mM in respect of EDTA and contains 1 % (w/v) of "Sarcosyl" (Na salt of N-lauryl-sarcosine), and which has a pH of 7.6, and the suspension is centrifuged at 20,000 rpm (40,000 G) for 20 minutes. The resulting precipitate is again taken up in the same buffer, and the mixture is warmed for 5 minutes at 65[deg]C and centrifuged again.The combined supernatants are made 0.3 M in respect of sodium acetate, 2.5 times the volume of ethanol is added and the mixture is kept at -20[deg]C. 2. Preparation of mRNA The RNA is separated from the ethanolic solution by centrifugation (20,000 rpm, (40,000 G), 30 minutes, 5[deg]C) and dissolved in a solution which is 0.5 M in respect of NaCI and 10 mM in respect of tris and which has a pH of 7.5. The resulting solution is charged onto an oligo-dTcellulose column (manufacturer: Collaborative Research, type 3), which has been equilibrated with the same buffer, and is eluted with 1 mM tris -solution of pH 7.6 or with distilled water. The elution of the RNA containing poly-A (mRNA) can be followed by measuring the extinction at 260 nm. The mRNA thus obtained is treated with sodium acetate until a final concentration of 0.3 M is reached, 2.5 times the volume of ethanol is added and the solution is kept at -20[deg]C. 3. Preparation of the cDNA a) Preparation of Single-stranded cDNA The conversion of the mRNA into the corresponding DNA (cDNA) is carried out with the aid of the enzyme reverse transcriptase. The incubation batch is: 50 mM in respect of tris, pH 8.3, 10 mM in respect of MgCI2, 30 mM in respect of 2-mercaptoethanol and 0.5 mM in respect of all 4 commonly occurring deoxyribonucleoside triphosphates (corresponding triphosphates of adenine, guanine, cytosine and thymidine), and contains 100 Microg/ml

Mannheim), about 100 Microg/ml of polyadenylated RNA and 800 units/ml of reverse transcriptase (manufacturer: Life Science Inc., St. Petersburg, USA). To follow the reaction, a deoxyribonucleoside triphosphate labeled by 32P in the a-position (specific activity: 50 Ci/mmole) can be added to the batch.The mixture is incubated for 60 minutes at 42[deg]C and the reaction is then stopped by adding 20 mM EDTA solution. The resulting solution is extracted with an equal volume of phenol saturated with water, the phenol is extracted by shaking with ether and , the ether residues are evaporated off with nitrogen. Non-incorporated deoxyribonucleoside triphosphates are separated off by column chromatography on Sephadex G50 in a solution which is 10 mM in respect of tris, pH 9.0, 100 mM in respect of NaCI and 1 mM in respect FEDTA. The eluted cDNA is precipitated by adding 0.3 M sodium acetate solution and 2.5 times the volume of ethanol. After centrifugation, the precipitate is taken up in 0.1 M NaOH solution and incubated for 20 minutes at 70[deg]C, or is incubated in 0.3 M NaOH solution overnight at room temperature. The mixture is brought to pH 7.6 with 1 M HCI and 1 M tris solution. b) Formation of Double-stranded cDNA (dsDNA) For the synthesis of the second DNA strand, again the enzyme reverse transcriptase can be used, or the enzyme polymerase I can be used. Formation of the dsDNA with Reverse Transcriptase The incubation batch (pH 8.3) is 50 mM in respect of tris, 10 mM in respect of MgCI2, 30 mM in respect of 2-mercaptoethanol and 0.5 mM in respect of the 4 above-mentioned deoxyribonucleoside triphosphates, and contains

transcriptase. The reaction is carried out for 120 minutes at 42[deg]C and is stopped by adding EDTA until a final concentration of 20 mM is reached. The phenol extraction and gel permeation chromatography on Sephadex G50 are then carried out as described above. Formation of the dsDNA with Polymerase I The incubation batch (pH 6.9) is 200 mM in respect of Hepes, (N-2-hydroxyethylpiperazino-N2-ethanesulphonic acid) 0.5 mM in respect of the 4 abovementioned deoxyribonucleoside triphosphates, 10 mM in respect of MgCI2, 30 mM in respect of 2-mercaptoethanol and 70 mM in respect of KCI. The reaction can be followed by adding deoxyribonucleotides, labeled by P (in the a-positions), to the incubation batch. The reaction is started with 800 units/ml of polymerase I and carried out for 2 hours at 15[deg]C. The reaction is stopped by adding a solution containing 0.1 % (w/v) of sodium dodecyl-sulfate and 100 Microg/ml of RNA. The resulting solution is extracted as described.The solution obtained after the extraction is charged onto a Sephadex G50 column and the chromatography is carried out with a solution which is 10 mM in respect of tris, pH 9.0, 100 mM in respect of NaCI and 1 mM in respect of EDTA. The eluted DNA can be determined by the radioactivity and is precipitated by adding 0.3 M sodium acetate solution and 2.5 times the volume of ethanol. The ethanol precipitate is taken up in a solution (pH 4.5) which is 300 mM in respect of NaCI, 30 mM in respect of sodium acetate and 3 mM in respect of ZnCI2, and 1,500 units ml of S1 nuclease (Boehringer Mannheim) are added. The reaction is stopped after one hour at 37[deg]C by adding EDTA until a final concentration of 20 mM is reached. Extraction with phenol and precipitation with ethanol are then carried out as described above. c) Preparation of cDNA in a "One-pot Process" As an alternative to the reactions described, the dsDNA can also be obtained from the mRNA in a "one-pot process'. In this process, the reaction of the reverse transcriptase is carried out as described, but 140 mM KCI solution is also added to the incubation batch. After the reaction has ended, the sample is heated at 100[deg]C for 4 minutes and the precipitate formed is centrifuged off. 0.4 M Hepes buffer (pH 6.9) is added to the supernatant in an equal volume, the 4 abovementioned deoxyribonucleotides being present in the buffer at a concentration of 0.5 mM. The reaction is carried out at 15[deg]C, as described, by adding 800 units/ml of polymerase I and is stopped by adding sodium dodecylsulfate and RNA. The procedure under 3.b), "Formation of the dsDNA with polymerase 1 ", is then followed. 4. Lengthening of the 3' Ends of cDNA with dCTP (Deoxycytosine Triphosphate) For incorporation into the plasmid, it is necessary to lengthen the 3' end of the DNA with one of the 4 abovementioned deoxynucleotides. Furthermore, the 3' ends of the cut plasmid are lengthened with the complementary deoxynucleotide. On bringing the DNA and the plasmid together, base pairing then takes place between the DNA and the plasmid. This recircularized molecule can be used for the transformation of bacteria; the bonds which are not yet closed are covalently linked by means of an enzyme system in the bacterium. For example, the 3' ends of the cDNA can be lengthened with dCMP residues (deoxycytosine monophosphate residues) and the plasmid can be lengthened with dGMP residues (deoxyguanidine monophosphate residues). On using these deoxynucleotides in the manner described, and on opening the plasmid by means of the restriction enzyme Pst I, a Pst I cleavage site is re-formed at every insertion site, after incorporation of the alien DNA, so that, after reproduction of the plasmid, the inserted DNA can easily be cut out again for further examination, using the restriction enzyme Pst I, again. The precipitate from the alcohol precipitation, after degradation with SI nuclease, is dissolved in an aqueous solution (pH 6.7) which is 30 mM in respect of tris, 1 mM in respect of CoCI2, 140 mM in respect of potassium cacodylate (cacodylic acid is dimethyl arsenous acid) and 150 MicroM in respect of dCTP, contains 100 Microg per ml of autoclaved gelatin hydrolysate and is 0.1 M in respect of dithioerythritol. P-labeled dCTP should be used to follow the reaction. The reaction is started by adding terminal deoxynucleotidyl transferase and carried out for 10 minutes at 37[deg]C. After the reaction time has elapsed, the sample is placed in ice and the number of incorporated dCMP residues is determined by radioactivity measurement in the precipitate which can be formed with trichloroacetic acid.In the reaction, about 10% of the nucleotides originally present should have been added; if this is not the case, the reaction can be continued by adding further enzyme. If too large a number of dCMP residues have been added, the resulting chain can be shortened with the aid of the enzyme S 1 nuclease. 5. Process for the Integration of DNA into a Plasmid A suitable circular plasmid, for example pBR 322, is cut with a restriction endonuclease which only recognizes one sequence on the plasmid. It is advantageous if this cleavage site lies behind a strong or inducible promotor and/or is located such that an antibiotic resistance is influenced. The plasmid converted to the linear form in this way is then lengthened at the 3' ends by one of the four deoxynucleotides. This is carried out, for example, in the following manner: 30,ug of plasmid are incubated with 50 units of Pst I restriction endonuclease in the presence of a solution which is 50 mM in respect of NaCI, 6 mM in respect of tris, 6 mM in respect of MgCI2 and 6 mM in respect of 2-mercaptoethanol and contains 0.1 mg/ml of autoclaved gelatin, hydrolysate for 40 minutes at 37[deg]C and at a pH value of 7.5. Extraction is then carried out with phenol and ether as described and the cut plasmid is precipitated with ethanol in the presence of 0.3 M sodium acetate solution. The lengthening of the 3' ends of the plasmid with dGTP (deoxyguanidine triphosphate) is carried out analogously to the lengthening of the cDNA with dCTP, described above. The lengthened plasmid-DNA is then mixed with the lengthened dscDNA in a solution (pH 8.0) which is 0.1 M in respect of NaCI, 10 mM in respect of tris and 1 mM in respect of EDTA, and the mixture is heated at 56[deg]C for 2 minutes, incubated at 42[deg]C for 2 hours and then cooled slowly to room temperature. The hybrid DNA obtained in this way is then used for the transformation of E. coli. 6. Cloning of the Hybrid Plasmid in E. coli The bacteria, for example E. coli X1776, are left to grow, at 37[deg]C in 50 ml of a customary nutrient medium, until an optical density of

sedimented, washed once with 10 mM tris solution of pH 7.5, then resuspended in 40 ml of a solution which is 75 mM in respect of CaCl2, 5 mM in respect of MgCI2 and 5 mM in respect of tris, and which has a pH of 8.0, and incubated for 20 minutes in ice. The cells are then sedimented and resuspended in 2 ml of solution which is 75 mM in respect of CaCI2, 5 mM in respect of MgCI2 and 5 mM in respect of tris and which has a pH of 8.0. 0.2 ml of this bacterial suspension is then mixed with 0.1 ml of hybrid DNA solution and the mixture is incubated on ice for 45 minutes. It is then warmed at 42[deg]C for 90 seconds and subsequently mixed with 0.2 ml of nutrient medium. 50 Microl 75 Microl of this suspension are then plated on agar containing tetracyclin and ampicillin and selection is made on the basis of antibiotic resistance. (Resistance to tetracyclines but not to ampicillins.) 7. Isolation of Strains which Produce Interferon-containing Proteins a) Immunological Detection With the aid of a test system developed by Broome and Gilbert (S. Broome and E. Gilbert, PNAS, 75, 2,746, 1978), the clones are examined for the expression of interferon-containing proteins. Clones which express interferoncontaining proteins can be detected, as a result of the binding of a radioactive antibody to these proteins, with subsequent autoradiography, by the fact that the X-ray film is darkened at this site: For this purpose, up to 50 clones per nitrocellulose filter are left to grow for 2 days at 37.C. The filters are then laid on an agar block which contains 1 mg/ml of lysozyme; a PVC film coated with interferon antibodies are applied to the bacterial colony and incubation then carried out for 2-3 hours at 4[deg]C. the PVC film is then incubated for 15 hours at 4[deg]C in a solution of

specific activity of the solution is about 1 x 106 cpm/ml. After washing and drying, the films are subjected to autoradiography. b) Biological Detection The clones are bred overnight in 50 ml of a conventional nutrient medium at 37[deg]C. The bacteria are then sedimented, washed twice with a cold solution which is 10 mM in respect of tris, pH 8.0, and 30 mM in respect of NaCI, and resuspended in a solution which is 30 mm in respect of tris, pH 8.0, and 1 mM in respect of EDTA and contains 20% of sucrose. The suspension is then shaken at room temperature for 10 minutes, the bacteria sedimented and resuspended in ice-cold H20 and the suspension is incubated in an ice bath for 10 minutes. The bacteria are then sedimented and the supernatant is tested in a virus inhibition test for the content of interferon-containing protein, in a manner which is in itself known. 8. Preparation of Interferon Protein The clones which show interferon activity in the various tests are bred in the conventional nutrient media and the bacteria are centrifuged off and extracted with aqueous buffers. The extraction solution contains the interferon protein.

Claims

1. A microbiologically prepared polypeptide, all or part of which comprises the amino acid sequence of human interferon.

2. A microbiologically prepared polypeptide as claimed in claim 1, all or part of which comprises the amino acid sequence of human fibroblast interferon.

3. A cDNA which codes for the amino acid sequence as claimed in claim 1 or claim 2, whenever prepared using mRNA obtained from human fibroblast cells.

4. A plasmid which comprises cDNA as claimed in claim 3.

5. Plasmid pBR 322 which comprises cDNA as claimed in claim 3.

6. A mciroorganism which comprises the genetic information required for the biosynthesis of a polypeptide as claimed in claim 1 or claim 2.

7. A microorganism as claimed in claim 6, being E. coli.

8. A microorganism as claimed in claim 7, wherein the E. coli is E. coli K 12 or E. coli X 1776.

9. A microorganism as claimed in any one of claims 6 to 8, wherein the genetic information comprises cDNA as claimed in claim 3.

10. A microorganism as claimed in any one of claims 6 to 8, wherein the genetic information comprises a plasmid as claimed in claim 4 or claim 5.

11. A process for the preparation of a microorganism as claimed in claim 10, which comprises introducing into the microorganism by transformation a plasmid as claimed in claim 4 or claim 5.

12. A process for the preparation of a polypeptide as claimed in claim 1 or claim 2, which comprises obtaining the genetic information for the biosynthesis of the polypeptide using fibroblast mRNA, incorporating the resulting gene into microorganisms in a manner which is in itself known, and selecting and culturing, in a manner which is in itself known, those microorganisms which produce this polypeptide.

13. A process for the preparation of a polypeptide as claimed in claim 1 or claim 2, which comprises obtaining from human fibroblasts mRNA for the polypeptide, producing double stranded cDNA therefrom, incorporating the cDNA into a plasmid, introducing the plasmid into microorganisms, and selecting and culturing those microorganisms that produce the polypeptide.

14. A process as claimed in claim 12 or claim 13, wherein the fibroblasts are induced with polyinosinic acid/polycytidylic acid in the presence of a protein synthesis inhibitor.

15. A process as claimed in claim 14, wherein the protein synthesis inhibitor is cycloheximide.

16. A process as claimed in any one of claims 13 to 15, wherein the plasmid is plasmid pBR 322.

17. A process as claimed in any one of claims 12 to 16, wherein the microorganism is E. coli.

18. A process as claimed in claim 17, wherein the microorganism is E. coli K 12 or E. coli X 1776.

19. A process as claimed in any one of claims 13 to 18, wherein the plasmid is introduced by transformation.

20. A process as claimed in any one of claims 12 to 19, wherein the RNA is isolated from the fibroblasts by arranging a caesium chloride-free lysate of the fibroblasts on an aqueous solution of caesium chloride, centrifuging the resulting system, and obtaining the RNA.

21. A process as claimed in claim 20, wherein the mRNA is isolated by affinity chromatography.

22. A process as claimed in claim 21, wherein the affinity chromatography is carried out on an oligo-dT-cellulose column.

23. A process as claimed in claim 12 or claim 13, carried out substantially as described in the Example herein.

24. A polypeptide as claimed in claim 1 or claim 2, whenever produced by a process as claimed in any one of claims 12 to 23.

25. A process for the preparation of RNA which comprises arranging a caesium chloride-free lysate as a layer on an aqueous caesium chloride solution, centrifuging the resulting system, and obtaining the RNA.

26. A process as claimed in claim 25, wherein the lysate is a lysate of human fibroblasts.

27. A process for the preparation of cDNA as claimed in claim 3, which comprises obtaining RNA from human fibroblasts, isolating the mRNA therefrom, and using the mRNA to prepare cDNA.

28.. A process as claimed in claim 27, wherein the RNA is obtained by a process as claimed in claim 25 or claim 26.

29. A process for the preparation of a plasmid as claimed in claim 4 or 5, which comprises introducing cDNA as claimed in claim 3 into a plasmid.

30. A process as claimed in claim 29, wherein the plasmid is plasmid pBR 322 and restriction endonuclease Pstl is used.