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    • 专利摘要:
    The present invention comprises novel DNA compounds that encode isopenicillin N synthetase and also comprises related methods, transformants, and polypeptides. The novel isopenicillin N synthetase-encoding DNA, together with its associated transcriptional and translational activating sequence, was isolated from Cephalosporium acremonium and cloned into an E. coli cloning vector. The isopenicillin N synthetase-encoding DNA has been used to construct novel E. coli expression vectors that drive expression of a stable, active, and novel isopenicillin N synthetase in E. coli. The intact C. acremonium isopenicillin N synthetase-encoding DNA and associated transcriptional and translational activating sequence have also been used to construct C. acremonium expression vectors that drive expression of the isopenicillin N synthetase in C. acremonium. The C. acremonium transcriptional and translational activating sequence has further been fused to a hygromycin phosphotransferase-encoding DNA segment and placed onto C. acremonium expression vectors. Useful derivatives of the novel compounds and vectors are also described.
    公开号:SU1623567A3
    申请号:SU884356444
    申请日:1988-09-19
    公开日:1991-01-23
    发明作者:Доминик Инголиа Томас；Виатт Квинер Стефен；Мэри Сэмсон Сьюллен；Лютеп Скэтруд Пол
    申请人:Эли Лилли Энд Компани (Фирма)；
    IPC主号:
    专利说明:

    The invention relates to bio-technology and can be used to obtain isophenicillin-M-synthetized.
    A new plasmid DNA was constructed to allow the expression of the isopenicillin M synthetase gene in Escherlchia coll cells.
    PRI me R. A. Plasmid isolation.
    Lyophilic E.col K12 JA221-plT335 is obtained from Northern Regional Research Laboratories, Peorla, Kfinols under the registration number NRRL B-15960. This Lyo Phil can be directly used as a culture in the proposed method.
    The E.colle K12 JA221 strain is incubated in 1 liter of 1 -bulon (10 gtrypton, YugMaC and 5 g yeast extract per 1 liter) containing 50 µg / ml ampicillin at 37 ° C until the optical density is reached at 590 nm (O.Dbe) - units of absorption, add 150 mg of chloramphenicol, incubate for another 16 hours.
    The cells are centrifuged at a speed of 6000 rpm for about 5 minutes at 4 ° C. The resulting supernatant is discarded, and the residue is washed in 40 ml of TE buffer; a (10 mM Tris-HCl, pH 7.5, 10 mM NaCl and 1 mM EDTA), then precipitated again. After the supernatant is re-merged, the cell residue is frozen in a bath with a mixture of dry ice-ethanol, thawed and repeatedly suspended in 10 ml of a solution of 25% sucrose and 50 mM EDTA. Then 1 ml of a solution containing 5 mg / ml of lysozyme, 3 ml of 0.25 M EDTA, pH 8.0 and tOO micron 1 10 mg / ml of PMAZA A is added, the solution is incubated on ice for 15 minutes. Then add 3 ml of the lysis solution obtained by mixing 3 ml of 10% Triton-X 100; 75 ml of 0.25M ED i A, pH 8; 15 ml of W Tris-HCl, pH 8.0 and 7 ml of water are mixed and incubated on ice for 15 minutes. Lysed cells are frozen in a dry ice-ethanol bath and then thawed.
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    Cell debris was removed from the solution by centrifuging at 25,000 rpm for 40 minutes in a SW 27 rotor (Beckman) and extracting with buffered phenol. After adding 30.44 g of CSCI and 1 ml (5 mg / ml) of ethidium bromide solution, the solution was adjusted to 40 ml , decant in a VTiSO cuvette ultra-plotrifuge (Beckman), centrifuged in a VTI50 rotor at 42000 rpm for 16h. The resulting plasmid band is visualized, irradiated with ultraviolet light, isolated, and then centrifuged at 50,000 rpm for 16 hours. All necessary changes in volume are made by adding TES buffer containing 0.761 g / ml CSCI. The plasmid band is again isolated, extracted with saturated saline isopropanol to remove ethidium bromide, diluted with 1: 3 TES buffer, two volumes of ethanol are added, and then incubated overnight at -20 ° C. Plasmid DNA was pelleted k by centrifuging the solution in an SS 34 rotor (Disrupted) for 15 minutes at 10,000 rpm,
    1 mg of plasmid DNA obtained in this way is suspended in 1 ml of TE buffer (10 mM Tris-HCl, pH 8.0 and 1 mM EDTA) and stored at -20 ° C.
    B, Construction of a plasmid.
    The lyophilic culture of E. col K12 RV308 / pCZ106 cells was used to inoculate 1 L of L-broth containing 50 µg / ml kanamycin, and then the inoculated broth was incubated at 25 ° C in an air shaker to O Dbeo between 0.5 and 0 absorption units. When these values reach an interval of 0.5 -1.0 units. absorption in optical density, the temperature is raised to 37 ° C and incubation is continued for 2-6 hours for uncontrolled replication.
    After incubation at 37 ° C for 2-6 hours, the cells are harvested, the plasmid pCZ106 DNA is isolated as in Example 1A. 5 mg of plasmid DcS2106 suspension is from-in 5 ml of TE buffer.
    To 25 µg of the obtained plasmid pCZ106 DNA was added 10 µg of a ten-fold BamHI reaction buffer containing 1.5 M NaCI: 60 mM Tris-HCl, pH 7.9, 60 mM MgCl2 and 1 mg / ml bovine albumin serum (BSA) , 50 items restrictase BamHI and 50 od restrictase N col in a volume of 5 μl and 55 μl of NaO. The resulting reaction solution is incubated at 37 ° C for 4 hours, after which the reaction is almost complete.
    N col-BamH, the reaction mixture is subjected to electrophoresis on a 1% agarose gel until a clear separation of the target
    1.6 kb N col-BamHi and 8.7 kb N col-N col fragments from other cleavage products, a 0.3 kb restriction fragment. Visualization of the electrophoresis-treated DNA is carried out by staining the gel in a dilute solution (0.5 µg / ml) of ethiidium bromide and exposing the tinted gel with a long-wave ultraviolet. After determining the position
    The 0 target fragments in the gel make a small gap in front of each of the target fragments and small pieces of the Schleicher and Schuell (Keene, NH 03431) NA-45 DEAE membrane are placed in each gap.
    5 When the electrophoresis is continued, the DNA binds to the DEAE membrane in a decorative manner, after the target fragments of the linkioioii. 1 with a DSAE membrane, these are stored and washed with a buffer containing 100 mM CA, 0.1 M EDTA, and 20 mM Tris-HCl, pH 8. Then each membrane is placed in a small vial and immersed in a buffer containing 1 M NaCI, 0, 1 mM PDTA and 20 mM trio-HCl, pH 8, are incubated
    5 at 65 ° C for 1 hour to remove DNA from memG: wounds After incubation at 65 ° C, the mono / basing buffer is collected, and the membrane is washed with.
    The DNA solution is adjusted so that the NaCl concentration is 0.25 M and three volumes of cold absolute ethanol are added. The resulting solutions are then mixed and left at (-70) ° C for 10-20 minutes. After cooling, the centrifugal solutions are boiled at 15,000 rpm for 15 minutes. After one more precipitation to remove the residual DNA salt, it is washed with ethanol, c1, pat, resuspended in 20 µl of TE buffer. The resulting purified fragments l.bkbNcol-BarnHlM 8.7 kb N col-Ncol t fiction fragments of the pCZ GG plasmid are separately dissolved in 25 µl of TE buffer and stored at (-20) ° C.
    25 μg of DNA g-plasmid ryspin5 restriction enzymes N col and BamHI, as described above. The N col-BamHI-digested DNA is placed on a 1% agarose gel and the target 1.5 kb of the N col BamH restriction fragment is isolated as described above. Approximately DMKI of the target fragment is obtained, the l / spendr is its comfort in 25 μl of TE buffer and stored. at 20 ° C
     μl 1.6 kb N col-Bamhl and 2.5, 7 kb
    5 N col-N col restriction fragments of the pCZ106 plasmid, are ligated with 5 µl 1,5kb of the N coi-BamHI restriction fragment of the plasmid pP335 until plasmid pi is obtained. The reaction volume is L f) µl and includes the indicated fragments
    DNA, 1.1 μl (100 units) of T4 DNA ligase, 3 μl of 10-fold ligation buffer (0.5 M Tris-HCl, pH 7.8; 100 mM MgCl2, 200 mM dithiomeritol EDTT); 100 mM ATP; and 1 mg / ml BSA / and 13.4 μl H2U. The reaction mixture is incubated at 15 ° C for 2 hours, after which the reaction is almost complete. The ligated DNA constitutes the target plasmid DNA.
    C. Designing E.coll K12 RV308 / pT337.
    A, Designing E.coll K12 RV308 / plT337,
    50 ml of E. coli K12 RV308 culture (NRRL B-15624) in L-broth are grown to O. DbEO 0.5 units of absorption. The culture is cooled on ice for 10 minutes, the cells are harvested by centrifugation. The cell pellet is resuspended in 25 ml of cold 100 mM CaCl2 and incubated on ice for 25 minutes. The cells are again precipitated by centrifugation, the pellet is resuspended in 2.5 ml of cold 100 mM CaCl2 and incubated on ice overnight. 200 μl of this cell suspension is mixed with the ligated DNA prepared as described above, incubated on ice for 20 minutes, and then the cells are harvested by centrifugation. The cell pellet is re-suspended in 1 ml of L-broth, and this suspension is incubated at 25 ° C for 1 hour. Aliquot portions of the cell mixture are placed on L-agar (L-broth with 15 g / L agar) containing 50 kmg / ml kanamycin, and these plates are incubated at 25 ° C. E.coll K12 RV308 / plT337 transformants are screened for kanamycin resistance and restriction enzyme analysis of their plasmid DNA.
    Some isolates of E. coli K12 RV308 / plT337 transformants are separately inoculated into 5 ml aliquots of L-broth containing 50 μg / mp kanamycin and incubated in an air shaker at 25 ° C until O is 5, 2 units, and then at 37 ° C for about 6 hours.
    After 6 hours, 1 ml of each cell culture is collected and centrifuged. Cell pellets are washed separately with 1 ml of 10 mM NaCI and then suspended in 1.0 ml of IPS extraction buffer (0.05 M Tris-HCl, pH 8.0, 0.01 M KCI and 0.01 MgS04). The cells are sonicated with pulses. 5.6 s, using microtips. The time between ultrasonic pulses is 60 seconds. The resulting mixture was kept in an ice-ethanol bath during this procedure. After sonication, the cell mixture is centrifuged to remove fragments, and then used directly in the assay.
    The control of isopenicillin-N synthesis was carried out in a volume of 500 µl. To start the reaction, 1.0 ml of a 1.4 mM d - / L -a aminoadipyl / -1-cysteinyl-0-valine solution and 3.75 mM DTT are left at room temperature for 30-60 minutes to bring any dimeric tripeptides into monomeric form. 50 µl of each of the following stock solutions are placed in small portions into control ampoules (sterile, glass, 13 x 100 mm ampoules): in a buffer of 500 mM Tris-HCl. pH 7.4, 100 mM
    5 KCI; 100 mM MgSCM; 1.0 mM FeSCM and 6.7 mM ascorbic acid. Then various amounts of extract diluted with water to a volume of 150 µl are added. About 100 µl of aliquots of the three peptide solution are then added to each vial; the addition of the tripeptide starts the reaction. Each vial is shaken, placed in a gyrotropic shaker bath while rotating at a speed of 250 rpm,
    5 at 25 ° C and incubated for 45 minutes.
    Then take two samples of 100 μl
    each, drip into the grooves of the plates for
    bioassay and the rest is 100 units.
    penicillinase B every reaction
    0, a mixture of dobavl from 5 µl 00 ccd) of rehydrated penicillinase-A and left for 5 min at room temperature and then 100 µl of each extract, co-isolated with penicillinase-A, placed in
    5 indentation on the bioassay plate. Such treatment with penicillinase-A is performed to verify that the areas on the bioassay plate are associated with the presence of penicillin, and not cefadostori0 or other impurities.
    The standard curve of penicillin -N is obtained by adding 0.5; 2.0; 5.0.10.0 and 20.0 μg of penicillin-N into the recess on the bioassay plates. The activity of penicillium 5-A-A is also checked, up to 5 µl of the enzyme preparation, to 200 µl of 0.2 µg / ml penicillin N.
    Plates for bioanalysis consist of K131 nutrient agar which is taken, a solution of 30.5 g of BBL Antibiotic Medium in 1 l of deionized water, boiling the solution, cooling to 70 ° C, and then keeping in an autoclave for 35 min at 121 ° C and pressure 1,055 kg / cm2. Then plate
    5 seeded with 4 ml of fresh overnight culture of Mlcrococcus luteus (ATCC 9341), a-per 700 ml of M.luteus agar is grown in K544 nutrient broth, which contains Dlfco peptone 5.0 g; Dlfco yeast extract 1.5 g; sodium chloride 3.5 g anhydrous dipotassium phosphate 3.7 g; monopotassium phosphate 1.3 g; Dlfco bovine extract 1.5 g in 1 liter of deionized water. The solution is brought to a boil, cooled to 25 ° C, brought to a pH of 7.0 with 1N. HCI or 1 and. NaOH, and then kept in an autoclave for 20 minutes at 121 ° C and a pressure of 1.055 kg / cm3 before use. Sowed agar is placed in plates of 100 x 15 mm in an amount of 15 ml of inoculated agar per plate. Grooves are obtained by suction with a 5 ml pipette; Each recess has a diameter of 10 mm.
    After the preparation of the plates, the samples are placed in these cavities and incubated at 37 ° C for 18 h. The results of the analysis determine the diameter of clean surfaces around each recess with the sample, which is formed because M.luteus cannot grow in the presence of penicillin.
    Although the linearity of the size measurement analysis “decreases markedly when the zone is larger than 21 mm, the results of the analysis show that E.caU K12 RV308 / pfT337 transformants express the activity of synthetase of i-penicillin-N, whereas the transformants of E. col K12 RV308 / pCZ106 do not do this.
    The enzyme produced by E. coli is significantly more stable than the synthetase of isopenicillin N obtained from Cephalotportun acremonlum. This higher stability is manifested in freeze and thaw experiments. C.acremonlum isopenicillin synthetaea is rapidly inactivated during freezing and thawing, but isopenicillin-N synthetase produced by E. col is sufficiently
    device freezing and thawing. Greater stability is associated with the difference in enzyme processing between C. acremonlum and E. col. For example, isopenicillin-N synthase, isolated from C. acremonlum, does not contain the first two amino terminal amino acid residues, methionine and glycine, which are present in the DNA encoding the activity of isopenicillin-N C. acremonlum synthetase and are also present in the proposed material produced E.soP.
    权利要求:
    Claims (1)
    [1]
    Invention Formula
    The method of producing recombinant plasmid DNA expressing isopenicillin-M-synthetase, consisting in that plasmids which are subjected to restriction endonuclease treatment are extracted from strains of bacteria Escherlchla coll K12 JA22./plT335 and E.coll K12 RV308 / pCZ106 obtaining N col-N col and N col-Bam HI fragments of the plasmid pCZ106 with a size of 7.8 kb and 1.5 kb, as well as a N col-BamHI fragment of a plasmid with a size of 1.5 kb; Ligirukp fragments obtained using DNA ligase in a buffer containing 50 mmol Gris-HC1, 10 mmol MgClz, 200 mmol DTT, 10 mmol ATP, bovine serum albumin, pH 7.8 at room temperature and 1 mg / ml DNA concentration about 2 hours, the obtained recombinant DNA transformed the E. coli RV308 bacterial strain with the selection of kanamycin-resistant clones, capable of expressing isopenicillin-L-synthetase activity, and isolating the target plasmid DNA.
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    法律状态:
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