SPECIFICATION
Microbiologically prepared polypeptide with the amino acid sequence of proinsulin of a primate, DNA and plasmids which code for this sequence, microorganisms which contain this genetic information, and processes for their preparation Human hormones are required to an increasing extent for therapy. Such hormones include inter alia insulin. Because it is impossible to obtain hormones in relatively large amounts from human organs, suitable techniques must be developed for producing these hormones outside the human organism.
The present invention indicates such a method. It relates to the isolation of nucleotide sequences which contain the genetic code for a specific protein, the synthesis of DNA with these specific nucleotide sequences, and the transfer of this DNA into a hot microorganism in which the replication and expression of this DNA takes place.
It has been proposed to prepare polypeptides and proteins by culturing bacteria carrying plasmids having genes which code for the desired polypeptide or protein. It is also known that it is possible to construct plasmids which cause the host by which they are carried to produce gene products which are not intrinsically characteristic of that host. It is also known that, by means of DNA recombination techniques, the genetic information for rat preproinsulin has been cloned in bacteria.
It has been possible to show that these bacteria express protein which possesses antigen determinants of insulin. Furthermore, it is known that the genetic information for the Achain and for the B-chain of human insulin has been cloned separately in bacteria. This was achieved by synthesizing the corresponding DNAs for the A-chain and for the B-chain de novo. It was proposed that the A- and Bchains produced by the bacteria could then be linked in vitro to form the insulin molecule.
None of these methods has led to a pharmaceutical agent which can be used in human therapy. In the case of rat proinsulin, the end product, namely rat insulin, is a foreign protein which leads to allergic reactions. In the case of human A- and B-chains, the properties of the B-chain, because of its tendency to polymerize, and the very poor yield in the combination of the A- and B-chains, are in opposition to the final aim, which is to produce human insulin.
The present invention therefore adopts a different method. It is known that monkey insulin and human insulin have an identical structure. If monkey proinsulin can be prepared, human insulin can be obtained therefrom by cleavage.
The present invention accordingly provides a microbially prepared polypeptide, all or part of which comprises the amino acid sequence of proinsulin of a primate.
The invention also provides cDNA which codes for a polypeptide as defined above, prepared using mRNA of a primate. The invention further provides a plasmid, especially plasmid pBR 322 which comprises the cDNA of the invention, and also provides a microorganisms, preferably E. coli, especially E. Coli K12 or E. Coli X 1776, which comprises a plasmid or the invention. The plasmid may be introduced into the microorganisms by transformation.
The invention provides, moreover, a process for the preparation of a polypeptide, all or part of which comprises the amino acid sequence of proinsulin of a primate, especially the amino acid sequence of the C-peptide of proinsulin of a primate, which comprises obtaining from a primate the mRNA for the polypeptide, producing double stranded DNA therefrom, incorporating the DNA 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.
This technique is applicable to the production of RNA from any source.
The following procedure can be adopted to achieve the aim according to the invention, the means illustrated being an example of several methods used.
Single cells are obtained from the pancreas of monkeys, preferably from Rhesus monkeys or cynomolgi, by proteolytic digestion, which must be controlled very carefully because of possible excessive degradation. The digestion is generally carried out using collagenase as the proteolytic agent. It has been found advantageous to carry out the digestion in the presence of mammalian serum, preferably bovine serum, and especially calf serum. It is preferably to use the serum in an amount of 20% or more of the total volume. The digestive provess is preferably repeated at least once, and the resulting mixture separated using centrifugation.
From these single cells, a layer rich in beta cells is obtained by density gradient centrifugation. This cell layer is denatured 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 lystate as a layer on an aqueous caesium chloride solution, and to centrifuge the resulting system. In contrast to methods proposed previously 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 RNA in the next reaction step.
After further precipitation, the mRNA is separated from the mixed RNAs by the use of an oligo-dT-cellulose column. The mRNA obtained is completed to give a 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) using reverse transcriptase or the eyzyme 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 RNA, for example with dCMP residues. A plasmid. for example pBR 322 or pBR 325. is cut with restriction nucleus 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 a microorganism, in particular into a bacterium, preferably E. coli, for example, E coli K 12 or 1776; the bonds which are not yet closed may be 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 nuclease used. Those clones which express insulin-containing proteins are selected with the aid of immunological tests. These clones are bred and the microorganism mass is centrifuged off, extracted and tested for insulin content. By this process, insulin values of more than one insulin unit ( = 1 IU) per liter of culture solution could be measured in a series of clones. This culture solution is used as the starting material for the preparation of monkey proinsulin and subsequently human insulin.
The following Examples illustrate the invention.
1. Process for the isolation of RNA a) The isolation of insulin-producing pancreas cells Pancreata are obtained under sterile conditions from primates, preferably Rhesus monkeys or cynomolgi, and. after removal of the skin and larger blood vessels. are mechanically comminuted by means of cutters. In this process. care must be taken to ensure that the tissue is kept cool whilst the work is being carried out, in order to prevent spontaneous digestion as a result of the pancreatin enzyme mixture contained therein. The comminuted tissue is washed several times with cooled mammalian serum, preferably calf serum, and then placed in small flasks and treated with 5 ml/g of a warmed sterile 0.125% strength (w/v) collagenase solution in PBS (phosphate buffered saline). The collagenase solution is always to be freshly prepared before the digestion.The simultaneous addition of 20% (v/v) or more of serum, preferably calf serum.
to the collagenase is decisive for the yield of cells from the pancreas. This mixture is stirred for 10 minutes and the whole supernatant is initially discarded. A collagenase/ serum mixture as above is then added again and the resulting mixture is stirred for about 6 to 10 minutes. This producess is repeated about 10 times and the digested product in each case is collected in a cooling vessel. The collected material with the enzymatically obtained cells is centrifuged at about 1,000 rpm (159 g) for 10 minutes in a cooling centrifuge, the supernatant is discarded and the cell deposit is resuspended in fresh Dulbecco's medium (see Bablanian et al., Virology 26 page 100, 1965) containing 1 g/liter of glucose and 20% (v/v) of calf serum. This centrifugation is repeated and the deposit which is then obtained is resuspended in 10 ml of Dulbecco's medium containing 1 5% (v/v) of added serum.
This cell material, which consists of a mixture of 8-cells, fibroblasts and epitheloid cells, is then separated via a density gradient, for example of Ficoll or Percoll. A Ficoll density gradient has proved to be best for this purpose. Thus, Ficoll 400 (Pharmacia) was dissolved in Dulbecco's medium without serum, and the solution was adjusted in a refractometer to a viscosity of 1 .38 or, with a spindle, to a density of 1 .16,filtered under sterile conditions through a membrane filter with a pore width of 0.45 Micro and kept at + 4[deg]C until used. From this Ficoll starting suspension, densities with a viscosity of 1,373, 1,370 and 1,3672 are established by adding Dulbecco's medium, and are arranged in layers on top of one another in a small centrifuge tube. The resuspended pancras cells are introduced on top and centrifuged for 10 minutes at + 4[deg]C and 3,200 rpm (2000 g).After the centrifugation, the individual bands are carefully obtained and free of residual Ficoll by wshing with Dulbecco's medium. The main mass of the insulin-producing beta -cells is concentrated on the uppermost layers with the lowest density and a viscosity of 1.3672.
b) Preparation of RNA The cells obtained are taken up in a ' denaturing medium" (4 M in respect of 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 solution is warmed at 80[deg]C for 5 minutes and immediately introduced into an ice bath.
This solution is then arranged in a layer on
hydroxyaminomethane (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 up in a solution which is 10 mM in respect of tris, 10 mM in respect of EDTA and of 1 % strength (w/v) in respect of "Sarcosyl" (Na salt of Nlauryl-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-dT-cellulose 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 (triphosphates of adenine, guanine, cytosine and thymidine),
ufacturer: Boehringer Mannheim), about 100 Microg/ml of polyadenylated RNA and 800 units/ml of reverse transcriptise (manufacturer: Life Science Inc., St. Petersburg, USA). To follow the reaction, a deoxyribonucleoside triphosphate labeled by P 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 of EDTA. 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 is 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 abovementioned deoxyribonucleoside triphosphates, and contains 50 Microg/ml of cDNA and 800 units/ml of reverse transcriptase. The reaction is carried out for 1 20 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-N'-2-ethanesulphonic acid) 0.5 mM in respect of the 4 abovementioned deoxyribonucleoside triphosphate, 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, labelled 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 chromato- graphy 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 SI 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 1 5[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 I", 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 re-circularized 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 Past I cleavage site is re-formed at every insertion site. after incorooration of the alien DNA. so that, after multiplication 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 to 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/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.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 SI 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 promoter and/or is located such that an antibiotic resistance is influenced.
The plasmid converted to the linear form in this way is lengthened at the 3' ends by one of the four deoxynucleotides.
This is carried out, for example, in the following manner:
30 Microg 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 MgCl2 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 ds cDNA in a solution (pH 8.0) which is 0.1 M in respect of NaCl, 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 plasma in E. coli The bacteria, for example E. coli X 1776, are left to grow, at 37[deg]C in 50 ml of a customary nutrient medium, until an optical density of A600 = 0.5-0.6 is reached, and they are sedimented, washed once with 10 mM tris solution of pH 7.5, then resuspended in 40 ml of a solution which was 75 mM in respect of CaCI2, 5 mM in respect of MgCI2 and 5 mM in respect of tris, and which had a pH of 8.0, and incubated for 20 minutes in ice. The cells then sedimented and resuspended in 2 ml of a solution which was 75.
mM in respect of CaCI2, 5 mM in respect of MgCI2 and 5 mM in respect of tris and which had 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.
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 insulincontaining proteins 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 insulin-containing proteins. Clones which express insulincontaining proteins can be detected, as a result of the binding of a radioactive antibody to these proteins, which 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[deg]C. The filters are then laid on an agar block which contained 1 mg/ml of lysozyme; a PVC film coated with insulin antibodies was applied to the bacterial colony and incubation was then carried out for 2-3 hours at 4[deg]C.
The PVC film was then incubated for 1 5 hours at 4[deg]C in a solution of iodine-labeled insulin antibodies. The specific activity of the solution was about 1 X 106 cpm/ml. After washing and drying, the films were subjected to autoradiography.
The clones reacting immunologically to insulin on the plate are bred in a conventional nutrient medium and the bacteria are centrifuged off, extracted and then tested for insulin content by radioimmunoassay. In this process, a number of clones show insulin values of more than 1 IU per liter of culture solution.