SU1724689A1 - Method for preparation of restriction endonuclease, recognizing and splitting the nucleotide sequence 5ъtgatca3ъ - Google Patents

Abstract

This invention relates to biotechnology and genetic engineering. The aim of the invention is to increase the yield and increase the activity of the target product. The method involves the use of Bacillus stearothermophilus (BKnM B-5209) as a producer strain, which has bacterial walls that are easily sonicated and contains only one restriction enzyme, which makes it possible to increase the yield of the target enzyme by 1.5 times, to facilitate the isolation and purification of the enzyme. The output of the enzyme is 65000 units / r of biomass, the activity is 15 units / μl. The restriction enzyme Bst 77, obtained by the proposed method, is a Bel I isospuomer and can completely replace it in all genetic engineering works. (Ls

Description

The invention relates to biotechnology and genetic engineering and concerns the production of a site-specific endonuclease capable of recognizing and cleaving the sequence of nucleotides b-TCATSA-3.

Restriction of such specificity can be used to study the primary structure of DNA, physical mapping of various genomes, expands the possibility of constructing hybrid molecules on a site, which is basically based on the widely used tetra-nucleotide 5 -CATS-3.

A known method for producing Bst GI restriction enzymes (recognition site b -TCATSA-3) and Bst Gil (recognition site 5 -CCA / T6C-3) from Bacillus stearothermophllus G3 strain.

The disadvantage of this method is the presence of two activities, which makes it difficult to obtain a pure enzyme that recognizes and cleaves the sequence of nucleotides 5 -TG PBX A-3

A known method for producing restrictase Bsp XII (recognition site s -TGATCA-3) and Bsp XI (recognition site 5 -ATCGAT-J), including the cultivation of the strain

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Bacillus sphaerlcus, chromatographic purification steps.

The disadvantage of this method is the low yield of the Bsp XII enzyme, which amounts to approximately 400 U / g of raw biomass. In addition, the presence of two enzymes complicates the purification procedure and the production of a targeted restriction enzyme without interfering contamination.

The closest to the present invention is the Bel I restriction enzyme production method that recognizes and cleaves the 5-TGATCA-3 site, including the use of the Bacillus caldolyticus producer strain, its cultivation for biomass production, multiple biomass disintegration, isolation and purification of the restriction enzyme by chromatographic in a way.

The disadvantages of this method are the low yield of the target enzyme (40000 units / r of raw biomass) and the low activity of the enzyme {1 unit / μl), as well as the strength of the cell wall of the strain makes it difficult to destroy the biomass and requires repeated disintegration.

The aim of the invention is to increase the yield and activity of the target product.

This goal is achieved in that according to the method for producing restrictase, Bacillus stearothermophilus strain (VKPM B-5209) is used as a producer.

The strain was isolated from thermal sources as a result of a search for producers of biologically active substances.

The resulting Bacillus stearothermophilus strain was deposited in the VKPM VNIIgenetiki under registration number B-5209, and the restriction strain produced by it is called Bst 77 I according to the generally accepted nomenclature.

The strain Bacillus stearothermophilus is characterized by the following features.

Morphological signs.

Cells are rod-shaped, straight or slightly curved (0.5-0.75) x (2-14) μm, motile. They form endospores of oval shape, terminal location, swollen relative to the size of the vegetative cell. Gram coloration is variable.

Cultural features.

Strain undemanding to growth factors, grows well on conventional nutrient media (SPA, MPA, BCH). On agar media, it forms small, round, non-pigmented colonies, transparent, shiny, with a smooth edge. The optimum growth temperature is 65 ° C, pH 7.0-7.2.

The culture is maintained by re-sowing in dense media, stored in a glycerol solution at -70 ° C or in a lyophilized state.

Physiological signs.

Does not restore nitrates, negative for lecithinase, in reaction with methyl red and Voges-Proskauer; does not hydrolyze starch, casein, gelatin, esculin; does not use malonate, citrate, does not grow when

NaCl concentrations of 5% and 0.001% lysozyme; vigorously forms acid from glucose, very weakly from arabinose, xylose and mannitol: it does not deminerate phenylalanine, does not release indole and hydrogen sulfide.

The strain is highly sensitive to antibiotics: penicillin, carbenicillin, ampicillin, gentamicin, methicillin, sulfathiazole, lincomycin; less sensitive to tetracycline, monomitsin, neomycin, oleandomycin, levomycetin, kanamycin, ristomycin, rifampicin; slightly sensitive to polymyxin; resistant to streptomycin.

For the cultivation of Bacillus

stearothermophilus B-5209 use medium of the following composition, g / l: peptone 10; yeast extract (Difco) 5; glucose 5; distilled water else. The cultivation is carried out at 65 ° C and intensive aeration until the stationary growth phase is reached.

The yield of the target enzyme 65000 units / g

raw biomass with an activity of 15 units / μl.

The difference of the proposed method from the known one is the use of a new, more productive strain of Bacillus stearothermophilus, exceeding in yield the known one more than 1.5 times, with a higher activity of the released

target enzyme, the ability to easily lyse upon ultrasonic disintegration,

Example. The strain is grown in a nutrient medium of the following composition, g / l:

peptone 10; yeast extract 5 (Difco); glucose 5; distilled water else. Cultivation was carried out at 65 ° C with aeration of 2 rpm, and under stirring, 150 rpm before the stationary growth phase.

Cells collected by centrifugation at 5000 g and a temperature of 4 C.

Biomass weighing 5 g is loaded into a glass beaker, 10 ml of 10 mM potassium phosphate buffer (pH 7.0) is added,

containing 1 mM EDTA, 7 mM 2-mercapto-ethanol (buffer A) with addition of 0.4 M sodium chloride, stirred using a magnetic stirrer to obtain a homogeneous suspension, add 6 mg of Lieozim and mix for 30 minutes.

The suspension is destroyed by an ultrasonic disintegrator at a resonant frequency of 22 kHz.

The voiced suspension is transferred to a beaker containing 50 ml of PEG-6000 (11.2%) and dextran G-500 (3.5%), placed on a magnetic stirrer, and with constant stirring, 4M Nad solution is added to a final concentration of 0.5 M. The mixture is stirred for 5 minutes.

To separate the phases, the suspension is centrifuged in a G-21 centrifuge at 20,000 rpm for 10 minutes. The resulting coarse extract (25 ml) is diluted twice with Au buffer applied to a 25 ml hydroxylapatite column. Impurity proteins are removed by washing with buffer A containing 0.2 M NaCl. The elution of the enzyme is carried out with a linear NaCI gradient of 0.01–0.25 M concentration with buffer A. The fractions containing the desired product are collected, the enzyme is diluted 1.5 times with 20 mM Tris-HCt (pH 7.5) containing 0.1 mM EDTA, 7 mM 2-mercaptoethanol, 10% glycerol (buffer B) and applied to a 30 ml heparin-rosehaph column. Impurity proteins are removed by washing with buffer B containing 0.2 M NaCl. The enzyme is eluted with a linear gradient of 0.2-1.0 M NaCI in buffer B. The fractions containing the desired product are combined and dialyzed against 50 mM Tris-HCl (pH 7.9) containing 1 mM EDTA, 7 mM 2- mercaptoethanol, 0.02% Triton X-100, 60% glycerol.

The output of the enzyme is 65000 units of act. / G of raw biomass, the activity of 15 units / μl. The drug does not contain impurities endonucleases. Per unit of activity, the minimum amount of enzyme necessary for complete cleavage of 1 µg of phage A DNA for 1 hour at 37 ° C is taken.

The proposed method, in comparison with the known, provides an increased yield of the target enzyme (more than 1.5 times), facilitates the isolation and purification of the enzyme from interfering restriction activity, since the strain used produces only one restriction enzyme, allows more restriction activity to be increased. than 10 times, and also facilitates the destruction of bacterial cells during ultrasonic disintegration.

Since restriction enzyme Bs-t 77 I has a recognition site 5 -TGATCA-3, identical to the Bel I restriction recognition site, it can replace the latter in all genetic engineering works.

Claims (1)

Invention Formula A method for producing a restriction endonuclease that recognizes and cleaves a sequence of nucleotides b -ATSATA-3 includes cultivating a producing microorganism of the genus Bacillus, disintegrating biomass, isolating and chromatographically purifying the enzyme, which is different in order to increase the yield and activity of the target product. as a producer, the Bacillus stearothermophilus VKPM B-5209 strain is used.