DE3102036A1 - METHOD FOR CLUTCHING A GENE WITH PROTEIN-ENCODING, IN PARTICULAR FOR THE PRODUCTION OF CHICKEN OVALBUMINE - Google Patents

Description

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TUC 3891

Dr. F / sm / January 22, 1981

THE UPJOHN COMPANY, Kalamazoo, Michiqan 49001, USA

Method for clapping a gene with protein coding, in particular for the production of chicken ovalbumin

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description

Numerous possible uses of recombinant DNA technology in terms of medical problems require the introduction of the biosynthesis necessary Proteins directing genes in host organisms. In most cases, a protein of interest will normally be synthesized in animal cells and not from yeast or other lower eukaryotes at home found. Although the cloning of a number of different animal genes with the information necessary to encode proteins has become possible, there have been few reports of these proteins clapping off in bacteria and other unicellular organisms. In some in E. coli expressed proteins is the human polypeptide hormone somatostatin (see K. Itakura, T. Hirose, R. Crea, A. D. Riggs, H.L. Heyneker, F. Bolivar and H. W. Boyer in "Science" Volume 198, pages 1056-1063, (1977)) on rat proinsulin and human insulin chains.

As is apparent from "Proceedings of the National Academy of Sciences" Volume 75, pages 5936 to 5940 (1978) succeeded recently the clapping or the expression of the chicken ovalbumin gene in E. coli HB101 by incorporating (melting) this gene in close to copy (transcriptional) and transmission or Deciphering (translational) starting areas. According to the invention is now the clapping or the expression of the chicken ovalbumin in yeast by incorporating (melting) it Gens succeeded in correct orientation relative to a copy start area.

As far as is known, there have been no reports of any foreign eukaryotic clapping or printing Gene in lower eukaryotes, e.g. B. yeast.

Using the DNA recombination method to be described below the chicken ovalbumin was attached to a S. cerevisiae copy control area melted. If one of the hybrid genes

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holding plasmid is housed in S. cerevisiae synthesized a protein identified as ovalbumin by immunoreactivity and polyacrylamide gel electrophoresis. The yeast-derived chicken ovalbumin is full-length (molecular weight: 43,000) and consists of approximately 1,500 molecules per cell. This is the first animal protein expressed or formed in yeast.

Quite generally, the invention consists in a method of clapping or printing out one for the host organism foreign gene coding for a protein in a suitable carrier, which is characterized in that the The removed gene is built into the carrier (fuse) near an existing copying start point and that the carrier is in a houses lower eukaryotic host.

Examples of other proteins to be considered are human serum albumin, human interferons, human antibodies, human insulin, Blood clotting factors, brain peptides, enzymes, virus antigens and proteins from plants.

The drawing shows the manufacturing process stages of the yeast plasmid pUC 1014 with built-in ovalbumin. Although the abbreviations used are customary and known to those skilled in the art, they are used to facilitate understanding redefined:

Restriction endonucleases: Sal I (in the examples) Tc: tetracycline resistance gene
amp: ampicillin resistance gene

Yeast his 3 gene: gene coding for an enzyme that is involved in biosynthesis of histidine in yeast is required T4 DNA ligase: enzyme encoded for this by bacteriophage T4 YEp: yeast episomal plasmid

pUC: Official name for a plasmid belonging to The Upjohn Company
pOV: plasmid ovalbumin
Ovalbumingen: Name for chicken ovalbumin eyes.

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The plasmids YEp 6, pOV 230 and pUC 1014 were in E. coli

Hosts in the permanent collection of Northern Regional Research

Laboratory, U.S. Department of Agriculture, Peoria, Illinois, USA deposited. Their deposit numbers are:

HBI01 NRRL B-1 1371 (DSM 1611) *

HB101 (pOV 230) NRRL B-11354 (DSM 1612) * HB101 (YEp 6) NRRL B-12093 (DSTi 1947) * HB101 (pUC 1014) NRRL B-12094 (DSM 1948) *

pUC 1014 was also found in a S. cerevisiae SHY 3 strain, which is a NIH accepted HV 2 host acts, deposited. The deposit numbers of these yeast deposits are:

S. cerevisiae SHY 3 strain: NRRL Y-12095 (DSM 1949) *

SHY 3 (pUC 1014): NRRL Y-12096 (DSM 1950) *

The plasmids mentioned are from the specified depository no later than the first publication of the reading ends Invention available. The handing over of the plasmids does not grant a license to any patent on the present Invention founded.

The YEp6 plasmid (K. Struhl, D. T. Stinchcomb, S. Scherer and R. W. Davis in "Proc. Nat. Acad. Jci." Volume 76, pages 1035 bis 1039 (1979)) contains an E. coli self-replication and an ampicillin resistance marker derived from pBR322 so that it can be obtained in E. coli. It also contains there is a yeast plasmid self-replication and a yeast His-3 gene, so that it can be obtained in his yeast auxotrophs. The YEp6 plasmid also has a unique SaI I restriction endonuclease site, which can be used to clone foreign DNA.

The structure of the YEp6-0valbumin melt plasmid püC 1014 proceeds in the manner shown in the drawing. The YEp6 plasmid is cut up with Sal I, whereupon the linear molecule formed is expediently treated with alkaline phosphatase

* Hintetlcqunqabezeichen in Deutsche Santnlunq of microorganisms of the Society for Biotechnological Research mbH, Grisebachstrasse 8, 3400 Göttinoen 13nfU9 / 057 £

to remove the 5'-phosphate groups at the ends of the molecule.

The pOV 230 plasinid contains almost the entire ovalbumin mRNA sequence including all information necessary to encode the amino acid sequence of chicken ovalbumin (See L. A. McReynolds, J. F. Catterall and B. W. O'Malley in "Gene" Vol. 2, pp. 217-231 (1977)). This Plasmid is cut with Sal I, with that treated with alkaline phosphatase, and cut with Sal I YEp6 linked and transformed into E. coli. Then take place a selection of the transformed host organisms on ampicillin plates and an analysis of their plasmid DNAs.

There are two possible orientations of ovalbuming relative to the YEp6 plasmid. These can be distinguished by Barn HI digestion of DNA and agarose gel electrophoresis.

Preparations of the plasmid DNAs are made by growing (growth) in E. coli and used to transform S. cerevisiae. His transformants are added to Minimal media plates selected under conditions where the His' parents cannot grow. After that will be the transformed host organisms are grown in broth and passed through a "French The extracts are lysed by means of a

I-solid phase radioimmunotests for their ovalbuminimmunoreak

investigated into activity.

This immuno-test shows that only one of the two possible orientations of ovalbumin is relative to the YEp6 plasmid is able to control the synthesis of ovalbumin in yeast. The hybrid plasmid with this orientation, namely pUC10i4, requires transcription or copy of the ovalbumin counterclockwise direction of the plasmid shown in the drawing. The other orientation requires a clockwise transcription or copy. Thus, the oval boiling is only clapped in yeast

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or printed out if it is in the YEp6 plasmid in the right Orientation is built in relative to a yeast copy start point.

A polyacrylamide gel electrophoresis of that produced in yeast Chicken ovalbumin shows that it migrates with an agility which is almost identical to the mobility of ovalbumin synthesized in the fallopian tubes of chickens. Some Differences in motility are due to incomplete modifications (after transfer) of that in yeast generated ovalbumin can be expected. Thus, the normal onset of transmission of the chicken gene becomes most likely exploited by the yeast transmission mechanism.

The following examples are intended to illustrate the invention in more detail. Unless otherwise stated, all mean Percentages “percentages by weight” and all quantities in the case of solvent mixtures “volumes”.

Example 1 (DNA extraction)

POV 230 (DSM 1612) is converted from pOV 230 (DSM 1612) using the method described by P. Guerry, D. J. LeBlanc and S. Falkow in "J. Bact." Volume 116, pages 1064-1066 (1973) described salt precipitation pOV 230 plasmid DNA isolated.

With an overnight broth culture of NRRL B-11354 L-broth containing 10 pg / ml tetracycline (cf. E. S. Lennox in "Virology" Volume 1, pages 190-206 (1955)). The cultures are kept at a temperature of Shaken vigorously at 37 ° C until the optical density at 600 nra is 0.8. After that, the plasmid copy number becomes 18 hours increased with chloramphenicol (250 μg / ml). After a one-off Wash in 50 mM Tris-HCl (pH 8.0) and 20 mM ethylenediaminetetraacetic acid the cells are in 33 ml of 25% sucrose solution in TE (10 mM Tris-HCl, pH: 8.0, 0.1 mM ethylenediaminetetraacetic acid) per liter of culture resuspended

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dated. Now 1 mg / ml lysozyme is added, whereupon the suspension Incubate for 5 min on ice. After adding 1/3 volume of 0.25 M ethylenediaminetetraacetic acid (pH value: 8.0) is incubated on ice for a further 5 min. The cells are by adding 10% sodium dodecyl sulfate solution in 37 mM Tris-HCl (pH: 8.0) with 67 mW ethylenediaminetetraacetic acid lysed to a final concentration of 1.3%. It is then incubated for 30 minutes at a temperature of 37.degree.

The chromosomal DNA is salted out by increasing the NaCl concentration brought to 1 M and then cooled to 4 ° C overnight will. The sodium dodecyl sulfate and chromosomal DNA are centrifuged at 17,000 χ g for 30 minutes removed at a temperature of 4 ° C. The supernatant liquid obtained in this way is precipitated using ethanol and pelletized and again solved in TE. This material is extracted twice with phenol and then with ether, with ethanol precipitated, pelleted and resuspended in TE.

The plasmid DNA is further purified by cesium chloride / ethidium bromide density gradient centrifugation. For this purpose, cesium chloride is dissolved in the DNA solution in an amount of 1: 1 (weight: volume), whereupon 550 μg / ml ethidium bromide are added. The gradients are centrifuged at about 100,000 χ g for about 40 hours. The plasmid DNA is removed from the gradient by needle puncture. The ethidium bromide is extracted with 1-butanol saturated _{with H 2 O.} After dialysis of the DNA in 10 mM Tris-HCl (pH: 8.0) and 0.1 mM ethylenediaminetetraacetic acid, ethanol is finally precipitated. The purified plasmid DNA is dissolved in 10 mM Tris-HCl (pH: 8.0) and 0.1 mM ethylenediaminetetraacetic acid.

If in the procedure described for obtaining pOV 230 DNA If the tetracycline is replaced by ampicillin, this process can also be used to produce YEp6 DNA and service pUC 1014 DNA in E. coli. Other plasmid DNAs

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you get in the context of this procedure, if you sign up for Maintenance of the plasmid in the culture of an appropriate selection, d. H. another antibiotic. The expert It shouldn't cause any difficulties either, the ones described Conditions for making plasmid DNA vary.

Example 2 (restriction endonuclease digests)

The Sal I digestion of YEp6 DNA and pOV 230 DNA obtained according to Example 1 is carried out in a reaction mixture with 6 mM Tris-HCl (pH value: 8.0), 6 mM MgCl ₂ , 150 mM NaCl, 6 mM β -Mercaptoethanol, 100 ug / ml of autoclaved gelatin, 80 ng / ml DNA and 80 units / ml Sal I restriction endonuclease. After incubation for 60 minutes at a temperature of 37 ^° C., the reaction mixture is extracted with phenol and then with ether and precipitated with ethanol. It should be noted that if a different carrier is used, a different restriction endonuclease may be required.

A person skilled in the art should have no difficulty in determining the reagent concentrations, to vary the substrates and enzymes as well as the reaction conditions.

Example 3 (treatment with alkaline phosphatase)

This measure is carried out with subordinate modifications essentially in the manner described by A. Ullrich,

J. Shine, J. Chirgwin, R. Pictet, E. Tischer, WJ Rutter and HM Goodman in Science Volume 196, pages 1313-1319 (1977). During 10 min 12 units / ml of bacterial alkaline phosphatase in 20 mM Tris-HCl are prepared at 7O ⁰ C (pH 8.0). After that,

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the 100 \ iq / ml obtained from in Example 2 excised with Sal I YEp6 DNA was added and a further 15 min is incubated at a temperature of 70 ⁰ C for. The reaction mixture is then extracted three times with phenol and then with ether and precipitated with ethanol. This measure represents an optional measure in the production of pUC 1014. However, the use of this measure leads to a higher ratio of pUC 1014 to parent YEp6 plasmid among the ampicillin-resistant transformants. Obtaining pUC 1014 becomes easier in this way.

Example 4 (T4 PNS Ligase)

A reaction mixture with 50 mM Tris-HCl (pH: 7.8), 10 InMMgCl ₂ , 20 mM dithiothreitol, 1 mM ATP, 30 μg / ml YEp6-DNA, 6 μg / ml of pOV 230 excised with Sal I and 15 units / ml T4 DNA ligase. After 16 hours of incubation at a temperature of 12.5 ° C is precipitated by means of ethanol, the reaction mixture, after which the pellet was resuspended in TCM (10 mM Tris'HCl, pH 8.0, 10 mM _CaCl2 / 10mM MgCl ₃ ) is solved.

A person skilled in the art should have no difficulty in determining the reagent concentrations, to vary the substrates and enzymes as well as the reaction conditions in a suitable manner.

Example 5 (transformation of E. coli)

120 ml of L-broth (1% tryptone, 0.5% yeast extract, 0.5% NaCl) are inoculated with an 18 h culture of HB101 (-JQSM 1611) and up to an optical density of 0.6 at 600 nm let grow. After washing in cold 10 mM MgSO ₄

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the cells are resuspended in 20 ml of cooled 50 mM CaCl ₂ solution for 15 min. The bacteria are then concentrated to 1/10 of this volume in CaCl ₂ and mixed with DNA linked according to Example 4 in a ratio of 2: 1 (V: V). After cooling the cell / DNA mixture for 15 minutes, the whole thing is subjected to a thermal shock at 42 ° C for 2 minutes, then brought to a temperature equilibrium for 10 minutes at room temperature and finally with 2 1/3 times the volume of the cells / DNA. Suspension L-broth added. The transformed cells are incubated for 1 hour in the broth at 37 ^° C., whereupon selective media (L-agar plus 20 μg / ml ampicillin) are inoculated with 200 μl / plate. ^{The plates are then incubated at a temperature of 37 °} C. for 48 hours to allow the transformants to grow. Although the transformation is of essential importance for the enlargement of biochemically constructed, newly combined DNA molecules, the person skilled in the art can vary the conditions to be observed without difficulty in order to achieve the desired result.

Example 6 (Transformation of NRRL Y-12095 to NRRL Y-12096)

The SHY 3 strain of Saccharomyces cerevisiae (ura trp leu his ~ ade ~) (DSM 1949) is transformed as follows: 20 ml of a log-phase culture, which up to an optical density at 600 nm of 2.0 (3 χ 10 ⁷ cells / ml) had been grown in YEPD broth (1% yeast extract, 2% peptone, 2% glucose), are pelleted and in 1/10 volume 0.9 M sorbitol, 50 mM KPO ₄ -puffer (pH- Value: 7.5) and 14 mM ß-mercaptoethanol resuspended. By addition of 1% Glusulase and 60 minutes of incubation at a temperature of 3O ⁰ C spheroplasts are formed. The spheroplasts are washed three times in 1 M sorbitol and then resuspended in 1/100 of the original culture volume of 1 M sorbitol, 10 mM Tris-HCl (pH value: 7.5) and 10 mM CaCl _2. Finally, pUC 1014 DNA is added to a final concentration of 20 μg / ml. After incubation for 5 minutes at room temperature,

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40% polyethylene glycol 4000, 10 mM Tris-HCl (pH 7.5) and 10 mM CaCl _{2 are} added to the 10 volumes, followed by another incubation for 10 minutes at room temperature. By overlaying minimal agar (0.7% yeast nitrogen base, 2% glucose, 2% agarose, supplemented with 20 ug / ml uracil, adenine and tryptophan and 30 ug / ml leu'cine) with 0.2 ml cells, which in 10 ml Melted (45 ° C.) regeneration medium (minimal medium with 1 M sorbitol, 2% YEPD and 3% agarose) His transformants are selected. The plates are incubated at a temperature of 28 ^{° C. for 5 to 6 days.}

Example 7 (yeast cell extract preparation)

Yeast cells that have been grown to an early stationary phase in 100 ml of minimal medium at 28 ° C are washed in Dulbecco's phosphate-buffered saline (Gibso) and in extraction buffer (1 mM Tris-HCl, pH: 7.4, 1 mM MgCl ₂ , 5 mM NaCl) in a ratio of 1: 1 (volume in ml: cell weight in g). The slurry is then passed through a French pressure cell at 1034 bar (15,000 psi) to lyse the cells.

Example 8 (solid phase radio immunity test)

This was done _ slightly modified - corresponding to that of Broome and Gilbert in "Proc. Nat. Acad. Sci." Volume 75, pages 2746-2749 (1978). Here, 0.2 mm thick polyvinyl chloride films with a diameter of 8 cm are _{allowed to float for 2 minutes at room temperature on 10 ml of 0.2 M NaHCO 3} (pH value: 9.2) with 600 μσ of the IgG fraction of anti-ovalbumin goat serum. Then the polyvinyl chloride film is turned over and on the other

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coated on the other side. Finally, the polyvinyl chloride film is washed with a solution (washing buffer) in the form of a phosphate-buffered saline solution with 0.5% normal rabbit serum and 0.1% bovine serum albumin. After washing, the films are brought into contact with the protein to be tested for the presence of immunoreactive ovalbumin. When cell lysates are to be tested, it is most convenient to place a lysate patch on an agarose gel matrix. After separation by electrophoresis, proteins can also be tested in a polyacrylamide gel matrix. The IgG-coated polyvinyl chloride is either brought into contact with the agarose matrix or Polyacrylamidgelmatrix and for several hours in a refrigerator (about 4 ⁰ C) incubated. After that, the

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Brought into contact with polyvinyl chloride I-labeled Antiovalbumin IgG and incubated overnight in the refrigerator. · After rinsing in wash buffer, the films using a commercially available film, and a commercially available intensifying screen at -70 ⁰ C are autoradiographed.

Example 9 (gene preparation and clonuna)

Structural genes coding for eukaryotic proteins are usually obtained using their purified messenger RNAs as starting material. Here, a complementary DNA (cDNA) copy of the messenger RNA is synthesized by enzymatic means and then made double-stranded by enzymatic means. This gene is then sent to an appropriate cloning vehicle bound, which is usually done by Poly dA: Poly dT attachment (see. D. A. Jackson, R. H. Symons and P. Berg in "Proc. Nat. Acad. Sci. Volume 69, pages 2904 bis 2909 (1972)), although this is not always required. The carrier containing the building is then turned into bacteria enlarged or reinforced. This measure was used for the production of the pOV 230 plasmid and for the production of Plasmids with globin genes and insulin genes performed.

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Example 10 (Other carriers, hosts and gene suppliers)

Examples of further carriers which can be used according to the invention are those capable of reproduction in yeast, e.g. B. YEp2, YEp4, YRp7 and YEp20 as well as vectors in others lower eukaryotic hosts for reproduction are capable.

Examples of further hosts for the carrier are any S. cerevisiae derivatives or other fungi. Of course the latter hosts must comply with NIH guidelines.

According to the invention, animal proteins of, for example Vertebrates, e.g. B. warm-blooded animals such as birds, e.g. B. chickens are contemplated. As already indicated, the invention extends quite generally to proteins that are foreign to the host organism. This also includes Proteins encoded by genes from plants or plant and animal viruses.

Example 11 (cleaning of ovalbumin)

The transformed yeast cells from Example 6 are allowed to grow up to an early stationary phase in a minimal medium (0.7% yeast nitrogen base, 2% glucose, which is supplemented with 20 mg / ml uracil, adenine and tryptophan and 30 μg / ml leucine) and then washed in Dulbecco's phosphate buffered saline. The cells containing ovalbumin are then resuspended in extraction buffer (1 mM Tris-HCl (pH value: 7.4), 1 mM MgCl ₂ , 5 mM NaCl) in a ratio of 1: 1 (volume in ml: cell weight in g). The slurry is passed through a French pressure cell at 1034 bar (15,000 psi) to lyse the cells. It is cleaned to obtain crystalline ovalbumin for bakeries or for research purposes

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in the method described by V. Shepherd and R. Montgomery in Methods in Carbohydrate Research "Volume VII (1976), edited by R. L. Whistler and J. W. BeMiller, Academic Press, New York, pages 172-174. Crystalline ovalbumin is from a wide variety of manufacturers available.

All carried out in connection with the invention Work was carried out in compliance with the NIH guidelines listed physical and biological safety requirements.

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Claims (20)

Claims 1. Procedure for clapping something alien to the host organism A gene encoding an animal protein in a group Carrier, characterized in that the gene in question is relatively in the correct orientation after removal to an existing copy start area in the carrier
incorporated and smuggled the carrier into a eukaryotic host. 2. The method according to claim 1, characterized in that one isolated the gene for animal protein from a vertebrate animal. 3. The method according to claim 2, characterized in that the gene for animal protein is isolated from a warm-blooded animal, 4. The method according to claim 3, characterized in that the gene for animal protein is isolated from a bird. 5. The method according to claim 4, characterized in that the gene for animal protein is isolated from a chicken. 6. The method according to claim 1, characterized in that a plasmid is used as the carrier. 7. The method according to claim 6, characterized in that YEp 6 is used as the plasmid. 8. A method for clapping the chicken ovalbuminbaugens in a suitable carrier, characterized in that the gene is built into the carrier after removal in the correct orientation relative to an existing copying start area
and introducing the carrier into a eukaryotic host. 130049 / 0S7A 9. The method according to claim 8, characterized in that a plasmid is used as the carrier. 10. The method according to claim 9, characterized in that YEp 6 is used as the plasmid. 11. SHY 3 (pUC 1014) (NRRL Y-12096-DSM 195Oj. 12. Plasmid pUC 1014. 13. The method according to claim 1, characterized in that a unicellular organism is used as the host. 14. The method according to claim 13, characterized in that one used a fungus as a unicellular organism. 15. The method according to claim 1, characterized in that the Kopierbeqinnbereich naturally in a unicellular organism sets an mRNA synthesis in motion. 16. The method according to claim 15, characterized in that the unicellular organism consists of a Pil2. 17. Process for the production of chicken ovalbumin, characterized in that rta'i man S. cerevisiae SHY 3 (oüC 1014) (NRRL Y-12096 -DSM 1950) under controlled conditions in one aqueous nutrient medium grows or lets grow. 18. A method for clapping a gene encoding a vegetable protein in a suitable carrier, characterized in that that after removal the gene in question is in the correct orientation relative to an existing copying start area built into the carrier and smuggled the carrier into a eukaryotic host. 19. A method for clapping a gene from a plant virus encoding a protein in an appropriate one Träqer, characterized in that the relevant 130 0 49/0574 310203a After removal, the gene is built into the carrier in the correct orientation relative to an existing copy start area and introducing the carrier into a eukaryotic host. 20. A method for clapping a gene from an animal virus encoding a protein in a suitable carrier, characterized in that, after removal, the gene in question is in the correct orientation relative to an existing one Incorporates copy start area into the carrier and smuggles the carrier into a eukaryotic host. 130049 / 057A