DE3605035A1 - PLASMID PWS101, METHOD FOR ITS DETERMINATION AND USE - Google Patents

Description

The invention relates to the new plasmid pWS101, which consists of a gene from the yeast Saccharomyces cerevisiae is obtained, as well as its use for the production of the enzyme L- Aspartates: 2-oxoglutarate aminotransferase E.C. 2.6.1.1.

This enzyme is used in clinical chemistry for manufacturing required by control sera. It has been out so far Pig hearts isolated. This type of production is complex and by using pig hearts as a raw material limited in the amount of product.

The invention is therefore based on the object Possibility of high expression of the enzyme L-aspartate: 2- oxoglutarate aminotransferase, E.C. 2.6.1.1. to create so that the production with little effort in time and costs as well as being easier and with a higher degree of purity.

The object is achieved according to the invention by the plasmid pWS101, characterized by the characteristics

• 1) reproducible in microorganisms of the species Saccharomyces cerevisiae and the species Escherichia coli,
• 2) carries the ASP5 gene from Saccharomyces cerevisiae, which for the enzyme L-aspartate: 2-oxoglutarate aminotransferase, E.C.2.6.1.1., Encoded, on
• 3) a "shuttle" vector with a centromere region (CEN4) from Saccharomyces cerevisiae (YCp50).

The microbial production of the Enzyme called allows. The procedural steps still required from plasmid pWS101 to the desired enzyme Standard techniques in molecular yeast genetics. The essentials in the manufacture of the enzyme is molecular Cloning of the yeast gene encoded for the enzyme Saccharomyces cerevisiae in one in yeast and the bacterium Escherichia coli replicable plasmid vector.  

The method according to the invention for producing the plasmid pWS101 is characterized by the steps

• a) Amplification and preparation of the in yeast and in Escherichia coli reproducible vector YCp50 from Escherichia coli strain 290A, cut with restriction endonuclease BamHI
• b) Chromosomal yeast DNA is with the restriction endonuclease Sau3A partially digested, the size fraction between 3 kilobase pairs (kbp) and 9 kbp thereafter in the cut vector from step a) inserted (ligated),
• c) with this preparation (ligase approach the Escherichia coli strain 290A in a manner known per se transformed,
• d) at least 10,000 transformants sensitive to tetracycline Step c) are combined and then the plasmid mixture prepared after propagation,
• e) with the mixture from step d), which is a yeast library a ura3-asp5 yeast mutant (WS8106-5B) transformed,
• f) Uracil-prototrophic and aspartate-prototrophic transformants from step e) are selected,
• g) with the extract of such a transformant becomes Escherichia coli 290A transformed and finally it looks like this Prepared yeast isolated plasmid.

Except for making the above. Enzyme can be the plasmid pWS101 also for the microbial production of amino acids be used.

The chromosomal yeast DNA of any aspartate prototroph Yeast strain is isolated and partially cleaned (Cryer, D.R., Eccleshall, R., and Marmur, J .; Meth. Cell. Biol. 12, (1975) page 39). On a small scale, conditions become for incomplete digestion with the restriction endonuclease Sau3A tested (Maniatis, T., Fritsch, E.F., and Sambrook, J .; Molecular Cloning, a Laboratory  Manual, (1982) Cold Spring Harbor Publ., New York), with two various suitable concentrations will be larger Cut amount of yeast DNA with Sau3A, the DNA is in one 0.5% agarose gel separated and stained with ethidium Bromide made visible. The size fraction between 3 and 9 kbp is cut out, the agarose pieces are cut in transferred a dialysis tube and electroeluted. The DNA is over disposable columns (from SCHLEICHER UND SCHUELL; Elutip) purified, precipitated with ethanol and in ligase Buffer added.

The vector used to construct the yeast library was in present special case the plasmid YCp50. This plasmid contains in addition to Escherichia coli and Saccharomyces cerevisiae recognized replication starting points (ORI or ARS) the E. coli genes for tetracycline or ampicillin resistance, the Yeast gene URA3 and the centromeric region of chromosome 4 (CEN4) (Kuo, C.L., and Campbell, J.L., Proc. Natl. Acad. Sci. USA 79, (1982), page 4243). Other vectors are also possible (Botstein, D., and Davis, R. W. In: The Molecular Biology of the Yeast Saccharomyces - Metabolism and Gene Expression, Strathern, J.N., Jones, E.W. and Broach, J.R. (Ed.), (1982) Cold Spring Harbor Laboratory Publ., New York, page 607). The vector used here is characterized by low copy number, but high mitotic Stability. The plasmid YCp50 is in E. coli amplified (chloramphenicol technique according to Maniatis, T., Fritsch, E.F., and Sambrook ,; J. Molecular Cloning, a Laboratory Manual, (1982), Cold Spring Harbor Publ., New York) and prepared after alkaline lysis (Birnboim, H. C., and Doly, J., Nucl. Acids Res. 7, (1979), page 1513); Alternative methods in Maniatis, T., Fritsch, E.F., and Sambrook, J .; Molecular Cloning, a Laboratory Manual, (1982) Cold Spring Harbor Publ., New York). It becomes complete with the restriction endonuclease BamH1 cut in the tetracycline resistance gene,  to avoid religion with alkaline phosphatase treated (Chaconas, G., and van de Sande, J. H .; Methods Enzymol. 65, (1980), page 75) and after ethanol precipitation taken up in ligase buffer.

The partially digested yeast DNA is treated in the so treated Vector ligated overnight at 7 ° C. With the ligation mixture a recombination-defective E. coli strain (290A, accession name DSM 3616, deposited as a plasmid-containing strain) transformed to ampicillin resistance (calcium chloride technology: Dagert, M., and Ehrlich, S. D .; Nature 275, (1978), page 104). On a small scale, conditions, the too high transformant frequencies and too high insertion frequencies lead, tested (high proportion of tetracycline sensitive Colonies among the transformants). With that verified ratios are transformed on a large scale, at least 10,000 transformants sensitive to tetracycline are combined by washing off the plates. Out some of the plasmids are prepared for checking. The size of the insertions is determined On average not less than 4 kbp. The so received Yeast gene bank is in the form of the transformant mixture -70 ° C (medium according to Beggs, J.D., Nature 275, (1978), page 104), for the preparation of the plasmids with Samples are inoculated and the plasmids are extracted from the Prepared cells of the stationary growth phase. With this Plasmid mixture becomes a uracil and aspartate auxotroph Yeast strain (WS8106-5B ura3-52 asp5, DSM 3617) after the Lithium acetate technology transformed (modified according to Ito, H., Fukuda, Y., Murata, K., and Kimura, A., J. Bacteriol. 153, (1983), p. 163); Alternative: spheroidal blasting method, according to Hinnen, A., Hicks, J.B., and Fink, G.R., Procl. Natl. Acad. Sci. USA 75, (1978), page 1929). From one of the uracil and aspartate protogrophic transformants obtained an extract is obtained (cells of the logarithmic Growth phase), plasmid DNA is enriched and prepared, E. coli strain 290A thus becomes ampicillin resistance  transformed and the plasmid of such a transformation is being prepared. The properties of this plasmid (pWS101), The asp5 mutation is complemented after renewed Yeast transformation confirmed. The plasmid is used with restriction enzymes mapped (figure).

The ASP5 gene codes for the enzyme glutamate oxaloacetate Transaminase (GOT, L-Aspartate: 2-oxoglutarate aminotransferase, E.C.2.6.1.1.). The activity of this enzyme is in Yeast extracts (preparation according to Sigurdson, D.C., Gaarder, M.E., and Livingston, D.E .; Mol. Gen. Genet. 183, (1981), Page 59) by a commercially available test (BOEHRINGER, Mannheim) easy to determine, the asp5- Mutant has no detectable GOT activity without a plasmid, however, it is transformed with the plasmid pWS101 significant GOT activity has been identified.

Due to the presence of this plasmid in a haploid Tribe that already has GOT activity becomes this activity increased by a factor of 2.

The strains of E. transformed with plasmid pWS101 coli (290A) and S. cerevisiae (WS8106-5B) are with the German Collection for Microorganisms, Göttingen, Grisebachstr. 8th, deposited under the number DSM 3616 or DSM 3617. Because the plasmid spontaneously with a certain probability is lost, so are the parent strains mentioned here available: without plasmid the strain 290A is tetracycline sensitive, the strain WS8106-5B needs uracil and aspartate.

The method according to the invention is described below with reference to Description of the steps of an implementation example in more detail explained.  

Implementation example:

• 1. Preparation of the DNA from 1 l of yeast culture (Saccharomyces cerevisiae) of the logarithmic growth phase.
• 2. Testing the conditions for partial digestion with the restriction endonuclease Sau3A (BOEHRINGER Mannheim).
• 3. Selected digestion conditions:
  • a) 200 µg DNA + 5.6 units Sau3A
  • b) 200 µg DNA + 2.8 units Sau3A
• incubated in 2 ml Sau3A buffer (20 mM TRIS-HCl, 50 mM NaCl, 6 mM MgCl ₂ , pH 7.5), 1 h at 37 ° C, stop the reaction by ice cooling and add EDTA (final concentration 2 mM ), then chloroform extraction, ethanol precipitation, absorption in TE (100 mM TRIS-HCl, 10 mM EDTA, pH 8.0).
• 4. union of the differently digested DNA samples, Separation of 200 µg thereof in 0.5% agarose gel (18th h, 35 V, 4 ° C, TRIS borate ethidium bromide buffer), Cut out the size fraction between 3 and 9 kbp (measured against λ-HindIII size standards, BOEHRINGER Mannheim), transfer into a dialysis tube, Electroelution (3 h, 100 V, of which 3 polarity reversed).
• 5. Removal of the buffer from the dialysis tube, purification and concentration of the DNA via disposable columns (SCHLEICHER UND SCHUELL Elutip), ethanol precipitation and absorption in ligase buffer (20 mM TRIS-HCl, 10 mM MgCl ₂ , 10 mM dithioerythritol, 0.6 mM ATP, pH 7.6).
• 6. Amplification of the vector plasmid YCp50 in the E. coli strain 290A (chloramphenicol technique), preparation from 1 l logarithmic culture (alkaline lysis), purification via cesium chloride density gradient centrifugation (36 h, 45,000 rpm), after dialysis and ethanol precipitation Uptake in BamH1 buffer (10 mM TRIS-HCl, 100 mM NaCl, 5 mM MgCl ₂ , 1 mM mercaptoethanol, pH 8.0).
• 7. Complete digestion of the plasmid under the restriction endonuclease BamH1 (BOEHRINGER Mannheim) at 37 ° C. overnight, extraction with chloroform, ethanol precipitation, absorption in 50 μl phosphatase buffer (50 mM TRIS-HCl, 1 mM MgCl ₂ , 0.1 mM ZnCl ₂ , 1 mM spermidine).
• 8. Testing the ligation conditions on a small scale, the following was then selected:
  3 µg YCp50 plasmid DNA + 3 µg partially digested yeast DNA + 5 units of T4 DNA ligase (RENNER), in 100 µl ligase buffer, incubation for 16 h at 7 ° C.
• 9. Transformation of E. coli strain 290A using this approach (10 µl samples each), about 70% of the ampicillin-resistant Transformants were sensitive to tetracyclines.
• 10. Trial preparation of the plasmids of 10 tetracycline sensitive Transformants, determination of the insertion size after EcoR1 digest (BOEHRINGER Mannheim), it was in the Average at 4.5 kbp.
• 11. 30,000 colonies were washed off the plates when Suspension stored at -70 ° C (medium according to Beggs, J.D. Nature 275, (1978), page 104). Growing samples thereof overnight on plates (approx. 30 plates LB with ampicillin), Preparation of the plasmid mixture (alkaline lysis, Cleaning the cesium chloride gradient).  
• 12. To transform the yeast strain WS8106-5B ( ade2-1 ura3-52 asp5 ) according to the lithium acetate method, 200 ml log culture was transformed with a total of 40 µg DNA, then plated on uracil- and aspartate-free medium (medium according to Sherman, F., Fing, GR, and Lawrence, CW, Methods in Yeast Genetics. Laboratory Manual, (1971), Cold Spring Harbor Laboratory, New York).
• 13. A transformant thus obtained was digested with zymolyase (SEKAGAKU KOGYO, via BAYER) an extract plasmid DNA was prepared by denaturation / Enrichment steps enriched, precipitated with ethanol and used to transform E. coli (290A). Plasmid preparation from a transformant, mapping the plasmid by restriction enzymes (Figure).
• 14. The possibility of complementing an asp5 mutation of yeast with this plasmid was confirmed by renewed yeast transformation. The following activities of the ASP5- encoded enzyme glutamate oxaloacetate aminotransferase were then measured in the yeast extract (BOEHRINGER Mannheim, GOT-Monotest):
  WS8106-5B (without plasmid) = 0, ie 2 units / g protein
  WS8106-5B (pWS101) = 53 units / g protein
  Protein determination against albumin standards (Lowry, OH, Rosenbrough, NJ, Farr, AL, and Randall, RJ, J. Biol. Chem. 193, (1951), page 265).

In a haploid strain that already has GOT activity, the activity is increased by the plasmid: regardless of as expected, this makes an absolute starting value Increase by a factor of 2.

Example:

WS8069 / 115 ( ASP5 , without plasmid) = 61 units / g protein

W8069 / 115 ( ASP5 , pWS101) = 126 units / g protein.

Figure: Preliminary restriction map of plasmid pWS101. Fragment length true to scale, gene location not exact (after Kuo, C.L. and Campbell, J.L., Proc. Natl. Acad. Sci. USA 79, (1982), page 4243). Restriction enzymes EcoR1, Sal1, Xho1 from BOEHRINGER Mannheim.

Claims (4)

1. Plasmid pWS101, characterized by the features

• 1) reproducible in microorganisms of the species Saccharomyces cerevisiae and the species Escherichia coli,
• 2) carries the ASP5 gene from Saccharomyces cerevisiae, which codes for the enzyme L-aspartate: 2-oxoglutarate aminotransferase, EC2.6.1.1
• 3) a "shuttle" vector with a Saccharomyces cerevisiae centrometer region (CEN4) (YCp50).

2. The method for producing the plasmid pWS101 according to claim 1, characterized by the steps

• a) Amplification and preparation of the vector YCp50, which can be propagated in yeast and in Escherichia coli, from the Escherichia coli strain 290A, cut with restriction endonuclease BamHI
• b) Chromosomal yeast DNA is partially digested with the restriction endonuclease Sau3A, the size fraction between 3 kilobase pairs (kbp) and 9 kbp is then inserted (ligated) into the cut vector from step a),
• c) with this preparation (ligase approach) the Escherichia coli strain 290A is transformed in a manner known per se,
• d) at least 10,000 tetracycline-sensitive transformants from step c) are combined and the plasmid mixture is then prepared after propagation,
• e) an ura3-asp5 yeast mutant (WS8106-5B) is transformed with the mixture from step d), which is a yeast library,
• f) uracil-prototrophic and aspartate-prototrophic transformants from step e) are selected,
• g) with the extract of such a transformant, Escherichia coli 290A is transformed and finally the plasmid thus isolated from yeast is prepared.

3. Use of the plasmid pWS101 for the production of the Enzyme L-Aspartate: 2-oxoglutarate aminotransferase E.C.2.6.1.1. (GOT) with the help of yeast. 4. Use of the plasmid pWS101 for microbial production of amino acids.