SU1294824A1 - Prism recombination plasmida and method for constructing same and e. coli c 600 p5 rm strain - producer of site-specific endonuclease p5 and ii - Google Patents

Abstract

The invention makes it possible to obtain an efficient strain-producing endonuclease PVU II. A recombinant plasmid pBP5RM DNA was constructed that determines the high level of synthesis of the Pvu II enzyme. The method of its construction consists in splitting the plasmid pBR322 DNA with the endonuclease Sal I, mixed with DNA from Proteus vulgaris hydrolyzed with the endonuclease. Xho I and the fragments are reunited with the DNA ligase of phage T4. The resulting mixture transform cells-E.col.and sown on selective medium with ampicillin. Plasmid ZpK was isolated from the clones obtained, hydrolyzed with the Pvu II endonuclease, and the resulting mixture was again transformed into E. coli cells. Ampicillin resistant clones are selected from which recombinant plasmids are isolated. The strain E.coli C600P5RM - producing site-specific endonuclease Pvu II was obtained by transformation of the plasmid PBP5RM into the strain E.coli C 600. 3 c. f-ly, 1 tab. with S (O y with 4 00 1C

Description

The invention relates to the field of}} ski6iological industry and molecular biology and is a recombinant plasmid DNA, determining the highly efficient synthesis of site-specific endonuclease, its method of construction and the strain carrying this recombinant plasmid-DNA DNA - producing a site-specific C-; Which endonucleases Pvu.II. ; The aim of the invention is the creation of an efficient producer strain of endonuclease Rlsch 11 suitable for the rapid purification of PTU II.

The goal is achieved by the fact that a recombinant design was constructed — shrcg-td rVRSH, which determines the high level of synthesis of the enzyme Pvu II.,

Sh

J5

The method is carried out as follows.

The plasmid DNA pBR 322 was cleaved with Sal I, and mixed with β-DNA. from Proteus viilgaris, hydrolyzed with the Xon I endonuclease, and fragments of the vi-f DNA-ligase of phage T4. The resulting mixture transform cells E, coll. and plated on ampicillin selective medium. Plasmid DNA was obtained from the clones obtained, hydrolyzed with endonuclease Pllt II, and the resulting mixture was again transformed into E. coli cells, and ampicolpine resistant clones were selected from which recombinant plasmids were isolated.

Strain producing site-specific endonuclease II, obtained

The method of constructing recombiate Q transformation of the plasmvda pVREM in

plasg-herzza is based on the hydrolysis of DNA from P. vulgarls with the endonuclease Kho I,; embedding the obtained fragments into vector DNA, followed by cloning in E. coli; and selection of recombinants containing the zononuc.lease gene PVU 11,

: As a shta1a producer of site-specific endonuclease Rlt II, the strain “coli C600P5Sh is used.

Ryokombinantny plasmid DNA rVRZRM size of 9000 base pairs contains the gene endonukleses. Pvu II and consists of the pBH322 DNA in which the DNA fragment obtained by hydrolysis of dac from / P, Tulgaris with the endonuclease Xho I is inserted by the Sal I site,

DNA fragment P, rulgaris has

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E.coli strain C 600, -. provides synthesis of endonuclease Rlt II in the amount of not less than 100,000 units. per 1 g of raw water; sa cells. Stagam t.coli С 600 Р5 Ш deposited in the All-Union collection of industrial microorganisms at BHHIi - Genetics under KB 3272,

The strain E.E.coli C 600 P5 RM, containing the plasmid pBPSRM, is characterized by the following: step 1 and features.

Morphological traits; cells of direct J. rod-shaped form 1.2 --- 1.6 X 2.0 X buO µm, a feat; 1 X with 5 peristrichum-b # 1 flagella, gram-negative.

Cultural features grow well on simple nutrient media. With growth on mono-peptone agar, colonies smooth J, round,. pressed against

sizes 4700 base pairs and contains Q brilliant, sulfur, e, the edge is even j.MyT-; gates for endonucleases C1A Is EcoPiI, when growing in liquid – 1x cf, m-m and Hind CTJ, the distance between k. ooty-peptone broth, In broth form mn are given in the table ,, evenly intensely cloudy.

Physiological and biological signs: grow within 10 - at optimum pH from b, ..., Many carbohydrates, alcohols, organic acids, in particular glucose, fructose, arabinose, lactose, and galactose are used as source carbon. As a source of nitrogen, mineral salt is used in ammonium and nitrate forms, in organic form — peptone ai OTHo acid, nitrates are reduced to nitrites.

Gelatinu not razzzhuyut. Urease a; coquity is not detected. Indole do not form.

Xho I. - Cla I0,9

Cla I - Hind III0,2

Hind III - Hind ill1,8

Hind III - Eco I, 6

EcoR I - Xho I 0.15

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55

five

The method is carried out as follows.

The plasmid DNA pBR 322 was cleaved with Sal I, and mixed with β-DNA. from Proteus viilgaris, hydrolyzed with the Xon I endonuclease, and fragments of the vi-f DNA-ligase of phage T4. The resulting mixture transform cells E, coll. and plated on ampicillin selective medium. Plasmid DNA was obtained from the clones obtained, hydrolyzed with endonuclease Pllt II, and the resulting mixture was again transformed into E. coli cells, and ampicolpine resistant clones were selected from which recombinant plasmids were isolated.

Strain producing site-specific endonuclease II, obtained

Q transformation of plasmvdy rVRZEM in

five

five

E.coli strain C 600, -. provides synthesis of endonuclease Rlt II in the amount of not less than 100,000 units. per 1 g of raw water; sa cells. Stagam t.coli С 600 Р5 Ш deposited in the All-Union collection of industrial microorganisms at BHHIi - Genetics under KB 3272,

The strain E.E.coli C 600 P5 RM, containing the plasmid pBPSRM, is characterized by the following: step 1 and features.

Morphological traits; cells of direct J. rod-shaped form 1.2 --- 1.6 X 2.0 X buO µm, a feat; 1 X with 5 peristrichum-b # 1 flagella, gram-negative.

Cultural features grow well on simple nutrient media. With growth on mono-peptone agar, colonies smooth J, round,. pressed against

Antibiotic resistance: exhibits ampicillin resistance.

PRI gER 1, Construction of recombinant DNA,

When constructing a recombinant plasmid pBP5RM, a fragment of P. vulgaris DNA is inserted into the recipient gshazmid. Illa bismus DNA pBR 322 was isolated from an E. coli RRI strain containing this plasmid 10 g of E. coli biomass RRI was suspended in 20 ml of 20% sucrose, 2 mM MgCl, 10 mg of lysozyme was added in 1 ml, and incubated at. 5 min, then 0.25 M EDTA (pH 8.0) was added to a final concentration of 40 mM, incubated for 10 min at 5 M NaCl to a concentration of 1 M, 10% Triton X-100 to 1% was added and left overnight at +4 C. Then the suspension was centrifuged at 100,000 g for 3 h, CsCl (0.8 g per 1 ml of solution) was added to the supernatant, centrifuged for 30 min at 5000 rpm, a film of proteins on the surface of the liquid was discarded, ethidium bromide was added to

0.4 mg / ml, after which the solution was centrifuged for 36 h at 50009 vol. min

on the rotor 50.2 Ti. The zone corresponding to the supercoiled DNA was taken, ethidium bromide was extracted with isopropanol; Plasmid DNA was precipitated with ethanol, the precipitate was dissolved in 0.5 M WaCl and further purified by DNA chromatography on a Biogel A-15 column. The fractions containing DNA were combined, the DNA was precipitated by adding 2 volumes of ethanol, the precipitate was separated by centrifugation, dissolved in and used as such in the future. The DNA of P. ailgaris was released after lysis of the cells with lysozyme,

. as described above, followed by phenolic deproteinization. After treatment with phenol, the aqueous phase containing DNA was dialyzed against 0.01 M atrium-citrate buffer (pH 7.5), incubated for 5 hours at 50 μg of pancreatic RNAase, then treated again with an equal volume of phenol, then the latter phases were removed by dialysis against 0.01 -M sodium citrate buffer. The obtained DNA preparations of P. vulgaris and PE h 322 were used to obtain recombinant plasmid pBP5RM.-DNA pBE 322 (1 µg) and P. vulgaris (3 µg), hydrolyzed in a buffer of 100 mM Tris-HC1 (pH 7.5), 10 mM MgCl.j, 2 mM dithymone, respectively, of the endonuclease Sal I and X1 – I I for 1 hour at 37 ° C, then the solutions were mixed, ATP was added to a concentration of 0.07 µM and 2 µl of phage T4 DNA ligase. The volume of the reaction mixture is 100 µl, the reaction is carried out at 16 h, then 1 h at 37 C.

P, R and ME 2, Obtaining producer strain PVU 11

To obtain the strain-producer of the Pvu II endonuclease, the mixture of recombinant DNA molecules obtained after treatment with a DNA ligase is transformed into the E. coli strain C 600, which allows it to be used as a host containing a low amount of non-insignificant nucleases.

compared with the original strain of P. vulgaris. 50 ml. Cultures of E.coji SOOP are grown to 2-3.10 cells / ml, precipitated at 6000 g for 15 min at 0 s, then washed twice with 25 ml of 0.1 M

CaC is resuspended in 0.5 ml

the same solution, to 12 ml of the DNA obtained after the ligase treatment, add 25 µl of competent cells. The mixture was kept for 20 minutes at 0 ° C, then 2 minutes at 42 ° C and 10, transferred into 1 ml of medium, incubated for 2 hours with aeration. The suspension is sown in 50 ml of medium containing 50 μg / ml ampicillin, grown to

late logarithmic phase, cells

They are precipitated by centrifugation and more: they are super-helix DNK of them, as

described in example 1, 5 μg of this DNA

process 1 hour at 37 ° C 10 units. Pvu IT endonucleases, then

transformation as described above. After transformation, cells are incubated in 1 ml of medium for 2 h and plated on C 50 μg / ml ampicillin. Transformants are selected for ampicillin resistance and tetracycline sensitivity. Clones satisfying these conditions are seeded in 5 ml of medium, grown to the late logarithmic phase,

Cells are harvested by centrifugation and the content of Pvu II endonuclease is tested in them.

Example 3. Testing of clones for the content of the Pvu II endonuclease was performed as follows. Clone cells grown in 5 ml of medium were suspended in 1 ml of 0.01 M potassium-phosphorus. Fat buffer (pH 7.0) containing

0.1 M NaCl, and sonicated for 3 min with ice cooling (amplitude 8 μm). Cell debris was precipitated at 40,000 g and Pvn II activity was determined in the supernatant, aliquots were diluted from 1/5 to 1/200 and 5 µl of each dilution was added to 20 µl of the reaction mixture containing 100 mM Tris-HCl (pH 7 , 8), 10 MgCl.j ,, 0.5 μg of DNA of phage 5 were incubated for 30 min at 37 37 Cj. The reaction products were analyzed by electrophoresis in O „gel with 8% agarose, the gel was stained with methyl bromide and was photographed under UV irradiation. . DNA fragmentation indicated the presence of the Pvu II endonase lease in the culture cells.

Clones that were found

to ampicillia, enables stable cell culture during the production of biomass.

5 Formula of invention

The recombinant pBPSRM plasmid, which encodes the biosynthesis of the site-specific endonu-Pase of Pvu II, has

0 the size of 9000 base pairs and consists of the following elements: EcoR I - Sal I - fragment of the plasmid pBR 322, spacing between sites of 650 base pairs, Xho I - Xho I fragment

5 DNA of Proteus vulgaris containing the Pvu TG endonuclease gene, and C1a I Hind III, Hind III, EcoR I sites, the distance between which (from the Xho I site) is 900, 200, 1800, 1600 and 150 pairs, the presence of endonuclease Rlga 11, you - 20 times respectively. Sal I - EcoR I

drop in 1 liter of liquid medium and determine the amount of PVU II endonuclease under standard conditions (hydrolysis 1 14kg phage DNA, 50 µl per I h) in them

The strain E.col i C 600 F5 RM contains an enzyme of at least 100,000 units per I g of biomass. A small amount of nonspecific nucleases makes it possible to test the amount of the Reti II endonuclease in the cell extract directly. Testing of Pvu II in the original P. vulgaris strain cannot be performed for non-specific nucleus

with the Sal I donuclease, the Proteus vul garis DNA is hydrolyzed with the Xho endonuclease, the obtained fragments are joined by the action of DNA ligay, this mixture is difficult to purify, and they are not easily purified.

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Plasma cells, valuable DNAs are processed with PVU IT, then the cells of E. colio are transformed from ampicillin resistant transformants, clones are selected, which do not allow the synthesis of Pvu 11 endonuclease from which the target plasmid is isolated.

th product.

Thus, the proposed 1–1 objects make it possible to obtain a strain of E. coli producing endonuclease II, the level of the enzyme content in the proposed strain is not less than 100,000 units per 1 g wet weight of cells, the endonuclease Pvu I is absent, the level of nonspecific,

The strain of Escherichia coli C 600 is much lower than that of the original strain, which exists in P5 RM (collection of the central museum of high purity organisms of the insulin enzyme preparation. Recombinant VNIIGenetics, collection plasmid pVREM, carrying the gel-number B-3272) producing site - Pvu II speciucleases and the resistant-digital endonuclease Pvu II,

Editor I. Egorova

Compiled by N, Kuzenksva

Tehred A. Kravchuk Proofreader, Cherni

562/27

Circulation 500 Subscription

VNIIPI USSR State Committee

for inventions and discoveries 113035, Moscow, Zh-35, Raushsk nab., 4/5

Production and printing company, Uzhgorod, st. Project, 4

to ampicillia, enables stable cell culture during the production of biomass.

Formula of invention

The recombinant pBPSRM plasmid, which encodes the biosynthesis of the site-specific endonu-Pase of Pvu II, has

size 9000 base pairs and consists of the following elements: EcoR I - Sal I - a fragment of plasmid pBR 322, the distance between sites is 650 base pairs, Xho I - Xho I fragment

Proteus vulgaris DNA containing the Pvu TG endonuclease gene and the C1a I Hind III, Hind III, EcoR I sites, the distance between which (from the Xho I site) are 900, 200, 1800, 1600 and 150 base pairs, respectively. Sal I - EcoR I

the pBR 322 plasmid fragment containing the pBR 322 plasmid replicon and the gene responsible for beta-lactamase synthesis (determining ampicillin resistance),

2 o A method for constructing a plasmid pBP5PME encoding the biosynthesis of a site-specific nucleonuclease, 3 concluded that the DNA of plasma 0 pBB 322 is cleaved en

with the Sal I donuclease, Proteus vulgaris DNA is hydrolyzed with the Xho I endonuclease, the resulting fragments are joined by the action of DNA ligay, this mixture 35 is transformed into E. coli, isolated from

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Plasma cells, valuable DNAs are processed with PVU IT, then the cells of E. colio are transformed from ampicillin resistant transformants, clones are selected, which do not allow the synthesis of Pvu 11 endonuclease from which the target plasmid is isolated.

  ,

Claims (2)

Claim The recombinant plasmid pBP5RM encoding the biosynthesis of the site-specific endonuclease Pvu II has a size of 9000 base pairs and consists of the following elements: EcoR I Sal I - a fragment of plasmid pBR 322, the distance between sites is 650 base pairs, Xho I - Xho I - DNA fragment Proteus vulgaris containing the Pvu IT. endonuclease gene, and Cla I Hind III, Hind III, EcoR I sites, the distances between which (from the Xho I site) are 900, 200, 1800, 1600 and 150 base pairs, respectively, Sal I - EcoR I A fragment of plasmid pBR 322 containing the replicon of plasmid pBR 322 and the gene responsible for the synthesis of beta-lactamase (defining ampicillin). '' 2. A method for constructing the plasmid pBP5RM encoding the biosynthesis of site-specific endonuclease, namely, that plaspBR 322 DNA is cleaved with Sal I endonuclease, Proteus vulgaris DNA is hydrolyzed with Xho I endonuclease, the fragments are joined by DNA ligase, and E. coli is transformed with this mixture, isolated cells with plasmid DNA, treated with Ρνύ II, then E. coli cells are transformed again. among the transformants resistant to ampicillin, 40 kp are selected, which ensure the synthesis of онνύ II endonuclease, from which the target recombinant plasmid is isolated. ι ... ¹ 3. The strain Escherichia coli C 600 P5 RM (collection of the Central Museum of Industrial Microorganisms of the Institute VNIIGenetika ”, collection number B-3272) - producer site - specific endonuclease Pvu II.