METHOD FOR INCREASING PLASMID YIELD

Abstract: This invention relates to an aqueous medium formulation which is capable of allowing the growth of a plasmid-producing host cell to a density greater than 30 at a reading of 600nm and, in addition, enhancing plasmid production beyond that which would be expected at the cell densities achieved. The medium of the present invention comprises a carbon source in the concentration range of about 20 to about 40 grams per liter of the water component of the medium.

Description: METHOD FOR INCREASING PLASMID YIELD Background of the Invention Increasing attention has been focused on the delivery of genes as therapeutic agents (i.e., gene therapy) for the treatment of gene-associated diseases. In particular, researchers have been studying the use of non-viral methods of delivery, due to safety concerns with using potentially infectious viruses. One of the most promising non-viral methods in gene therapy is the use of cationic lipids as a delivery vehicle (Felger, Proc. Natl. Acad. Sci., 84:7413- 7417, 1987). The cationic lipids bind with negatively charged DNA and facilitate entry of the DNA into target cells. Successful delivery of genes via lipids into airway epithelia of rodents {Hyde, Nature, 362:250-255, 1993) have been reported. The gene of interest is generally incorporated into plasmids which can be produced and isolated from bacterial cells. Cationic lipids are generally synthesized chemically or isolated from natural sources using various methods known in the art. If lipid delivery of genes proves to be effective, huge quantities of plasmids will be required for future gene therapy. However, current methods for producing genes may impede progress in this field. Currently there are very few reports on plasmid yield enhancement. One method involves the addition of a protein inhibiting drug, chloramphenicol, into a host culture (Maniatis et al. Molecular Cloning, A Laboratory Manual, 2d, Cold Spring Harbor Laboratory Press, 1989). This approach does not increase plasmid yield significantly and, in addition, chloramphenicol is toxic to humans (AHFS Drug Information, American Society of Hospital Pharmacists, 1994). In another method, the plasmid producing cells are grown in a fed batch mode (Hofman et al., J. Basic Microbiol., 30(1):37-41, 1990). While this approach can produce large quantities of plasmids, production yield is only proportional to cell mass. A need exists to develop economically viable methods for producing plasmids on a large scale to meet the increasing demand. Summary of the Invention This invention relates to an aqueous medium formulation which is capable of allowing the growth of a plasmid-producing host cell to an optical density greater than 30 at 600nm and, in addition, enhancing plasmid production beyond that which would be expected at the cell densities achieved. The medium of the present invention comprises a carbon source in the concentration range of about 20 to about 40 grams per liter of the water component of the medium. In another aspect, the present invention relates to a method of increasing a plasmid-producing prokaryotic cell to a density greater than an O.D. reading of 30 at 600nm and of enhancing the plasmid production of a prokaryotic host cell by culturing the prokaryotic host cell in an aqueous medium with the composition comprising a carbon source in the range as stated above. Brief Description of the Drawing The Figure is a graph of the actual plasmid yield obtained from a culture of prokaryotic cells using the medium of the present invention versus the plasmid yield that would be expected given the cell density. Detailed Description of the Invention This invention is based upon the discovery that using an aqueous medium formulation with a high concentration of a carbon source increases prokaryotic cell density and plasmid production beyond that achieved using the prokaryotic medium formulations disclosed previously. The medium of the present invention comprises a carbon source in the concentration range of about 10 to about 40 grams. Suitable carbon sources include glucose, mannose, galactose, maltose and glycerol. Glucose in a concentration of about 30 grams per liter of the water component of the medium is preferred. It is believed that the medium of the present invention is suitable for any prokaryotic cell. Suitable cells include those belonging to the genuses Norcardia, Bacillus, Corynebacterium and Escherichia. It should be noted that prokaryotic media formulations in addition to requiring a carbon source need to provide a nitrogen source, a trace metal mixture, a complex vitamin source (although some prokaryotes can produce their own) and an inorganic salt mixture in order to sustain prokaryotic cells in culture. Suitable nitrogen sources for use in the medium of the present invention include ammonium chloride, primatone CLT, casein enzymatic hydrolysate or any other protein digest. In a preferred embodiment the medium contains about 10.0 g of ammonium chloride, 10.0 g primatone CLT, 20.0 g casein enzymatic hydrolysate each per liter of the water component of the medium. A suitable trace metal mixture for use in the medium of the present invention would include a mixture of sodium molybdate, manganese sulfate, cupric chloride, cobalt chloride, boric acid and zinc chloride. In a preferred embodiment the mixtures contains 4.0 mg sodium molybdate, 20.0 mg manganese sulfate, 2.0 mg cupric chloride, 8 mg cobalt chloride, 1.0 mg boric acid and 4 mg zinc chloride. A suitable inorganic salt mixture would include magnesium sulfate, potassium sulfate and ferrous sulfate. In a preferred embodiment the mixtures contains 0.4 g magnesium sulfate, 0.2 g potassium sulfate and 12.7 mg ferrous sulfate A suitable complex vitamin source for use in the medium of the present invention would include a yeast extract such as Bacto yeast extract. Any method for growing prokaryotic cells known to those skilled in the art are suitable for using the medium formulation of the present invention in the method of present invention. See Gerhardt, P, et al., Manual of Methods for General Bacteriology, Published by American Society for Microbiology, 1981. Any method for determining optical density known to those skilled in the art are suitable in the method of the present invention (see Gerhardt, P,) EXEMPLIFICATION Materials and Methods: Frozen E. coli cells containing the appropriate plasmid were used to inoculate 100 ml of Luria-Bertani Medium (LB) (components of which are listed in Table 1 below) in 2 X 500ml shake flasks. The culture was incubated at 37+0.5°C for 15-18 hours on an orbital shaker (New Brunswick Scientific, New Brunswick, NJ) at 150-250 rpm. Table 1 : LB FORMULATION # Components grams/liter of H2O 1. Bacto Yeast Extract 5 2. Bacto tryptone 1 0 3. sodium chloride 1 0 The seed culture was used to inoculate a 20 liter fermentor containing 15 liters of a medium components of which are listed in Table 2 below: Table 2: THE MEDIA FORMULATION OF THE PRESENT INVENTION # Components A m o u nt/l iter of H 2 0 1 . glucose 30.0 g 2. Bacto Yeast Extract 20.0 g 3. ammonium chloride 10.0 g 4. potassium phosphate, monobasic 1.5 g 5. magnesium sulfate.7H2O 0.4 g 6. potassium sulfate 0.2 g 7. Primatone CLT 10.0 g 8. Casein Enzymatic Hydrolysate 20.0 g 9. Ferrous Sulfate. 7H2O 12.7 mg 10. calcium chloride.2H2θ 4.0 mg 1 1 . sodium molybdate.2H2O 4.0 mg 12. manganese sulfate 20 mg 13. cupric chloride.2H2O 2.0 mg 14. cobalt chloride.6H2O 8.0 mg 15. boric acid 1.0 mg 16. zinc chloride 4.0 mg 17. Pluoronic 25-R-2 (BASF) 0.67 ml 18. am picil lin 0.1 g The culture was allowed to grow for 15 hours under the conditions listed in Table 3 below: Table 3. GROWTH CONDITIONS # Condition Measure 1. agitation 800 rpm 2. aeration 1.0+0.1 vvm 3. temperature 37 * C 4. pH controlled at 7.0+0.1 Culture growth was determined by monitoring optical density with a cell density probe (Cerex Co., Jamesville, ND) at 600 nm until all glucose had been depleted and the optical density had reached 35- 40. We allowed the culture to continue until plasmid concentration stopped increasing in around 4 to 8 hours after glucose was depleted. The final O.D. 600nm reading was 60+5. Harvest of the culture was by centrifugation. The cells were stored at -70° to -80°C until purification. Cell Growth and Dried Cell Weight Measurement Cell growth was determined by monitoring optical density with a cell density probe (Cerex) at 600 nm. Dried cell weight was determined by centrifuging (5000 x g for 10 min. at 4°C 50 ml. culture, washing the cell pellet three times with deionized water and dried in a 60°C (6-7 hours) hot air over until a constant weight is obtained. The dry cell weight in expressed as gram per liter of culture. Glucose Measurement Glucose concentration was measured with a biolyzer (Kodak, Rochester, NY) using the procedure provided by Kodak. Plasmid Purification The cells were suspended in Tris/EDTA/RNase A, pH 8 and lysed with NaOH + SDS. Cell debris, proteins and chromosomal DNA were precipitated with 2.5-3 M potassium acetate. The plasmid was then purified, using Nucleobond (The Nest Group, Inc., Southboro, MA) plasmid purification column following the instructions provided. Plasmid Analysis The concentration of plasmid was estimated by measuring absorption at 260 nm. The purity of the plasmid is determined by: 1 ) ratio of A260/A280, in an acceptable range of 1.8-2.0, Spectophometer (Hewlett Packard, Germany) 2) visual inspection of an ethidium bromide stained agarose gel after electrophoresis, the absence of RNA and genomic DNA bands on the gel indicates good quality. The quality of the plasmid is determined by restriction maps, in vitro transformation of mammalian cells and assaying for the gene product (Maniatis et al.).

Claims: CLAIMS 1 . An aqueous medium formulation for the enhancement of plasmid production of a prokaryotic host cell, the medium comprising a carbon source in the concentration range of about 20 to about 40 grams per liter of the water component of the medium. 2. The medium formulation of claim 1 , wherein the carbon source is a sugar. 3. The medium formulation of claim 2, wherein the sugar is glucose. 4. A method of enhancing plasmid production from a prokaryotic host cell, comprising culturing the prokaryotic host cell in an aqueous medium formulation wherein the medium comprises a carbon source in the concentration range of about 20 to about 40 grams per liter of the water component of the medium. 5. The method of claim 4, wherein the carbon source is a sugar. 6. The medium formulation of claim 5, wherein the sugar is glucose. 7. In a process for producing plasmids from a prokaryotic host cell wherein the improvement comprises culturing the prokaryotic host cell in an aqueous medium formulation containing at least 20 grams of a carbon source per liter of the water component of the medium. 8. The improvement of claim 7, wherein the carbon source is a sugar. 9. The medium formulation of claim 8, wherein the sugar is glucose. 10. A method for enhancing the production of plasmids from a prokaryotic host cell, comprising: a) culturing the prokaryotic host cell in an aqueous medium containing at least 20 grams of a carbon source per liter of the water component of the medium; b) allowing production of the plasmids in the prokaryotic host cell; c) collecting the medium containing the prokaryotic cell in step b; d) isolating the host cell from the medium; e) lysing the host cell to release the plasmids; and f ) purifying the plasmids released from the host cell in step e. 1 1 . The method of claim 10, wherein the carbon source is a sugar. 12. The medium formulation of claim 11 , wherein the sugar is glucose. 13. An aqueous medium formulation which increases prokaryotic cell culture density to a O.D. reading greater than 30 at 600nm, the medium comprising a carbon source in the concentration range of about 20 to 40 grams per liter of the water component of the medium. 14. The prokaryotic medium formulation of claim 13, wherein the carbon source is a sugar. 15. The medium formulation of claim 14, wherein the sugar is glucose. 16. A method of increasing prokaryotic cell density culture to a O.D. reading greater than 30 at 600nm, comprising culturing a prokaryotic host cell in an aqueous medium formulation comprising a carbon source in the concentration range of about 20 to about 40 grams per liter of the water component of the medium. 17. The method of claim 16, wherein the carbon source is a sugar. 18. The medium formulation of claim 17, wherein the sugar is glucose.

Inventors: GOODRICK JASON C, WAN NICK C, GOODRICK, JASON, C., WAN, NICK, C.
Applicants: GENZYME CORP, GENZYME CORPORATION
Application year: 1995
Main classification: C12N1
IPC codes: C12N1, C12N15, C12R1