SU1458388A1 - Method of producing endonuclease-restrictases capable of recognizing and splitting nucleotide sequences - Google Patents

Abstract

This invention relates to the microbiological industry, to methods for producing an enzyme that specifically cleaves DaC. Restriction enzymes can be used to study the primary structure of DNA and in i genetic engineering. The purpose of the invention is to simplify the method of obtaining and increasing the yield of the target products. The method consists in using a strain of Haemophilus influenzae RFL I (BiaiM B-4035) as a producer of restriction enzymes. Cultivation is carried out under conditions of aeration on a nutrient medium until the optical density of the cell suspension is 1.8-2.5 oe. Cells were disrupted by sonication, centrifuged and allowed purification of enzymes from cell-free extract obtained by chro matografii phosphocellulose, followed by purification of restriction enzyme Hin And Blue Sepharose and geparinsefaroze and chromium matograficheskoy purification Rest riktazy Hin III on DEAE-cellulose and geparinsefaroze. When using the method, high yields of highly purified restriction enzymes are obtained. Hin II and Hin III

Description

I

The invention relates to the microbiological industry, to the production of enzymes, in particular to a method for producing restriction enzymes.

The aim of the invention is to determine the method of preparation and increase the yield of the target products.

The strain of Haemophilus influenzae RFL I obtained from the Central research Institute of vaccines and serums to them. I.I. Mechnikov and stored in the All-Union Collection of Industrial Microorganisms of the Institute of the All-Russian Scientific-Research Institute of Genetics under the number VKPM B-4035.

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Used strain Haemophilus

influenzae RFL I has the following

morphological, cultural and

|) iziologicheskie signs. Cells

coccobacilli. Gram-negative,

motionless, capsule-free; requires

for growth X and Y factors; on dense

mediums form transparent small

colonies; hemolysis is missing .. Strain

1 forms urease, breaks down xylose;

forms an indole, does not have a /, galactoside, and does not utilize ornithine (refers to the second Killian biotype on an agar brain extract

and the hearts of the calf after 24 hours of incubation in a thermostat form small, round, V with even edges of the colony. The colonies are smooth, shiny, agar does not grow together. The culture is not propagated in moc-peptone broth. Optimal growth temperature

.

carried out as follows

Way-

Example. Production of restriction enzymes Hin 1 I and Hin 1 II. From Haemophilus influenzae RFL I.

Sequence of operations. I. Cultivation of strain and polybiomass. , Isolation and purification of restriction II pelvis:

, 2.

Cell disruption Chromatography on phosphocellulose.

3.1. Purification of Hin 1 I reetriktase:

3.1.1. Fractionation of proteins on blue sepharose.

3.1.2. Chromatography on heparin-Sepharose.

3.2. Cleaning restrictase Hin 1 II:

3.2.1. Chromatography on DEAE cellulose ..

3.2.2. Chromatography on heparin-sepharose.

1. To obtain the biomass of Haemophilus influenzae RFL I, the following equipment is used: a laboratory fermenter, Biolafite (France) ,. pH meter, thermostat, autoclave, circular rocking chair, SF-26 spectrophotometer, flow-through unit: 1 CELL type (German Federal District), laminar box.

For multiplication and cultivation of the strain, use is made of a brain brain extract (Brain Heart Infusion Difco, with the addition of X and Y factors. Nutrient medium is in its composition

contains

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3.7% extract of the brain and the heart of the calf, 0.02% nicotinamide adenine dinucleotide (NAD) and 0.1% hemin, pH 7.0. Heart-brain extract is sterilized at 1 atm for 20 minutes, hemin solution - at 70 ° C for one hour, solution NAD - ultrafiltration.

The strain of H. influenzae RFL I is maintained in a lyophilized form at + 4 ° C. The culture protective environment is stored separately as follows: the sucrose solution and the gelatins are sterilized. At 0.9 atm for 30 minutes twice a day and before preparing the bacterial suspensions in the so prepared sterile solution under aseptic conditions add the same amount of bovine serum.

The recovery of the culture in two tubes with a nutrient medium (cardio-brain extract, NAD and hemin) and two tubes with peptone broth (to control the purity of the culture) are transferred to the lyophilized culture. The tubes are incubated for 18 h at 37 ° C, then the bacteriological loop of the culture is re-seeded in two tubes with nutrient medium and medium with peptone broth and placed on a circular rocking chair at 80-100 rpm. After 18-20 hours cultivation was carried out the third passage - for direct seeding of the fermenter: 0.25-0.30 ml of the thus obtained cell suspension was added to two flasks with 0.25 liters in each 40 medium. The inoculum is grown on a circularly controlled rocking chair at 200 rpm for 18-20 hours. The optical density of the suspension or Rock at a wavelength of 540 nm is 3.0-3.5 o. The resulting inoculum is seeded in a laboratory biolafit fermenter containing 10 liters of nutrient medium1 1. The culture of H. influenzae RFL I is grown at. pH 7.0-7.2 with a stirring speed in the first hour of cultivation at 150 rpm and then up to 250 rpm and also in the supply of sterile air of 3 liters / min. in the first hour of growth of the tours with a gradual increase to 5 liters / min. 6th hour of growth. The target pH during the culture is maintained by the addition of saturated sodium bicarbonate solution.

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During the cultivation period, the troughs are periodically taken and the optical density of the cell suspension is measured. The cultivation of the strain is carried out until the late logarithmic growth phase — the optical density of the cell suspension is 1.8-2.5 oe. (wavelength 540 nm).

The culture fluid is cooled to + 4 ° C and centrifuged in a flow-through centrifuge of the CEPA type at 2.5-10 rev / min. The resulting biomass is stored at -20 ° C. The yield of crude biomass 2.5-,.) 3.5 g / l of culture fluid.

Ii. The following equipment is used to isolate and purify the restraints: ultrasonic disintegrator UZDN-2T, Beckman J21C centrifuge, Minikoldlab refrigeration chamber, chromatographic columns, thermostat, electrophoresis apparatus, universal power source.

The DNA of phage lmbda was isolated by a known method (Salto N., Miura K.I. Biochem. Biophys Acta, 1963, 72, 619-629).

For cell disruption and chromatography, a buffer solution (B) is used: 10 mM potassium phosphate buffer pH 7.4, containing 10 mM 2-mercaptozanol.

The activity of restriction enzymes is tested by the method of hydrolysis of DNA of phage lmbda followed. separation of the obtained fragments by agarose gel electrophoresis. The reaction mixture for DNA hydrolysis of phage lambda with the Hin 1 I restriction enzyme contains 10 mM Tris-HCl, pH 8.5, 25 mM NaCl, 5 mM MgCl, 100 µg / ml bovine serum albumin, 2 µg of phage lambda DNA in 40 µl of the mixture and 5 μl of enzyme. The reaction is carried out at 37 ° C for 5 minutes. The reaction mixture for the hydrolysis of DIiK phage lambda with the restriction enzyme Hin.1 II contains 10 mM Tris-HC pH 8, B, 50 mM NaCl, 10 MM.MgCli, 100 µg / ml bovine serum albumin, 2 µg DNA of phage lambda in 40 μl of the mixture and 5 μl of the enzyme.

The reaction is carried out at 10 min.

The electrophoresis was carried out in a 1 M sodium borate buffer, pH 8.2, 2 mM EDTA for 2 hours at a voltage of 101 V and a current of 100 mA. Ethidium bromide stained (1 μg / ml) gels are viewed in UV light.

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ABOUT

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The standard unit of activity is the amount of enzyme that, under optimal conditions, fully cleaves 1 µg of phage lambda DNA in 1 h.

All operations for the isolation and purification of the enzyme are carried out at.

1..The destruction of cells. 30 g biog. the masses obtained by cultivating N. influenzae RFL I are suspended in 100 ml of buffer B containing 0.1 M NaCl, sonicated on a UZDNNT disintegrator for 15 minutes and centrifuged at 20,000 rpm in a Beckman F-21C centrifuge, rotor JA- 20.

2. Chromatography on phosphocellulose. The cell-free extract obtained was applied at a rate of 30 ml / h on a 2.5–15 cm column with phosphocellulose equilibrated with buffer B containing 0.1 M NaCl. The column was washed with 150 ml of the same buffer and eluted with a linear gradient of NaCl from 0 to 1 M in 500 ml of buffer B. Separately collected fractions eluted at 0.25–0.36 M NaCl containing most of the restriction activity - PS Hin 1 I, and the fractions eluted at 0.58-0.7 M, containing the activity of restrictase Hin 1 II. Further, the purification of restriction enzymes Hin 1 I and Hin 1 II is carried out separately.

3.1. Cleaning restrictase Hin 1 I.

3.1.1. Fractionation of proteins on blue sepharose.

The fractions containing the restriction enzyme activity Hin 1 I are combined and dialyzed for 16 hours against buffer B containing 0.2 M NaCl. The fermentation solution after dialysis at a rate of 20 ml / h is applied to a l-cm column with blue sepharose, equilibrated with buffer B containing 0.2 M NaCI, and washed with 60 ml of the same buffer. Collect the elute containing the major part of the restriction activity (fraction I).

3.1.2. Chromatography on heparin-Sepharose.

Fraction 1 at a rate of 20 ml / h was applied to a 1-cm column with heparin-sepharose, equilibrated with buffer B containing 0.2 M NaCl, washed with the same buffer (30 ml) and eluted with a linear gradient of NaCl from 0.2 to 0 , 6 M in 240 ml of buffer B. Fractions, zluiruemye

/ 1458388

1; | and 0.3-0.4 M NaCl, are also combined with these restrictases.

The menth preparation is concentrated by dialysis against buffer B containing and, 1 M NaCl, 0.1 mM EDTA and 50% -gly-Cerin (by volume). The enzyme preparation is stored at -20 ° C. Of the one biomass frame, 15,000 units of Hin 1 I restriction enzyme activity are obtained.

3.2. Purification of restrictase Hin 1 II. Yu

: 3.2.1. Chromatography on DEAE-cel-Lulose.

i Fractions containing the activity of 11striktazy Hin 1 II, are combined and e: ializiru1ot for 16 h against buffer B. After dialysis, the solution of the enzyme is centrifuged at 20,000 rpm

electrophoregrams coincide with electrophoregrams obtained after DNA processing of phage lmbda only by Hin 1I restriction enzyme. This indicates that these restrictases are isoschizomarae, i.e. 5 GPuCGPyC-3 nucleotide sequence is cleaved to confirm the recognizable sequence of nucleotides and to determine the cleavage site, partial hydrolysis of 5-P labeled Hin 1 I pBR322 DNA fragments was performed. 15 two-dimensional division of the resulting

hydrolyzate by acetyl cellulose electrophoresis and homochromatography in a thin layer of DEAE cellulose resulted in a fingerprint from which it was found.

on the Beckman Zh-21C centrifuge, rotor) | СА-20.

The resulting supernatant is quickly coupled with a homologous sequence of 20 ml / h and applied to the column. The cleavage area is a 1.5 cm x 15 cm with DEAE-cellulose, suspended with buffer B, washed with TIM with the same buffer (80 ml) and eluted with a linear a gradient of NaC1 from O, O to, 5 M in 280 ml of buffer B. Fractions, fluted at 0.04-0.12 M NaCl, Combine; they contain the main part of the restriction activity (fraction 1 | 1 and 1).

3.2.2. Chromatography on. Heparin- (bepharose.,

Fraction 1 at a rate of 20 ml / h with a niocHT per column of size G, cm C with heparin-sepharose equilibrated with buffer B containing 0.1 M NaCl, washed with 30 ml of the same buffer and eluted with a linear NaCl concentration gradient of 0.1 to 1, 0 M in 120 ml of buffer B, Fractions eluted at 0.68–0.8 M NaClj are combined And the enzyme preparation is concentrated by dialysis against buffer B containing 0.1 M NaCl, 0.1 mM. EDTA and 50% glycerol (by volume) The enzyme preparation is stored at. From one gram of biomass, 1000 units of Hin II restrictase activity are obtained.

Determination of sequences

ranucleotides 5 -CG (T / C) G-3. Therefore, restriction enzyme Hin 1 I spreads a recognizable area between

25 second and third bases, fragments with 5 protruding sticky ends:

5 -GPuCGPyC - 3; 3 CPyGCPuG-5,

30 To determine the nucleotide sequence recognized by the restriction enzyme Hin 1 II, DNA of phage 01 74, fd and plasmids pBR322 with a known primary structure were used. They were subjected to restriction enzyme Hin 1 II and the obtained data on the number and length of fragments were compared with tabular ones (Fucbs C. et al Gene (1978), 4, 1-23 Fuchs C. et al Gene, 1980, 35740 370). Comparison of these results made it possible to discover that the nucleotide sequence 5 -C.ATG, recognized by the restriction enzyme Nla III, has the same frequency of occurrence.

45 substrates, like Hin 1 II. From this, it was suggested that the Hin 1 II restriction enzyme has identical substrate specificity with restriction Nla III. Eyelet confirmation

The nucleotides recognized by restriction enzymes, the specificity of the Hin I and Hin 1 II restriction enzymes under study, were obtained after determining the sequence of nucleotides recognized by the restriction enzyme Hin 1 I, DNA of phage lambda was used. This DNA was cleaved with the restriction enzyme Hin 1 I, with the restriction enzyme A ha II (which recognizes VL cleaves the sequence of the nucleorection of the substrate cleavage site. For this, a self-complementary synthetic decadvocy-55 synucleotide containing the investigated enzyme was used:

tido W -GPuCGFyC-3) and together

5 -GCG

3 -CGC

GCG.

these restrictases

In all cases

electrophoregrams coincide with electrophoregrams obtained after DNA processing of phage lmbda only by Hin 1I restriction enzyme. This indicates that these restrictases are isoschizomarae, i.e. The nucleotide sequence of 5 GPuCGPyC-3 is cleaved. To confirm the recognizable sequence of nucleotides and determine the cleavage site, partial hydrolysis of 5-P labeled Hin 1 I pBR322 DNA fragments was performed. Two-dimensional division of the resulting

hydrolyzate by acetyl cellulose electrophoresis and homochromatography in a thin layer of DEAE cellulose resulted in a fingerprint from which it was found.

that the homologous sequence at the cleavage site is 5-CG (T / C) G-3 tetranucleotides. Therefore, restriction enzyme Hin 1 I spreads a recognizable area between

second and third bases, fragments with 5 protruding sticky ends:

5 -GPuCGPyC - 3; 3 CPyGCPuG-5,

To determine the nucleotide sequence recognized by the restriction enzyme Hin 1 II, DNA of phages 01 74, fd and plasmids pBR322 with a known primary structure were used. They were treated with the restriction enzyme Hin 1 II and the obtained data on the number and length of the fragments were compared with tabular ones (Fucbs C. et al Gene (1978), 4, 1-23; Fuchs C. et al Gene, 1980, 10, 357370). A comparison of these results revealed that the nucleotide sequence 5 -C.ATG, recognized by the restriction enzyme Nla III, has the same frequency of occurrence on the substrates used as Hin 1 II. From this, it was suggested that the restriction enzyme Hin 1 II has identical substrate specificity with the restriction enzyme Nla III. A conclusive confirmation of the specificity of the restriction enzyme under investigation was obtained after determining the location of the cleavage of the substrate. For this, a self-complementary synthetic decadvococ 55 synucleotide containing the site recognized by the enzyme under study was used:

5 -GCG

3 -CGC

GCG.

After the introduction of the end label, the double-stranded substrate was treated with the restriction enzyme Hin 1 II and the labeled products of the restriction enzyme reaction were analyzed in a thin layer of DEAE-cellulose. In a parallel track, a partial 3-exonuclease hydrolyzate of the same labeled decanucleotide, which is part of the duplex under study, was analyzed. The only labeled cleavage product was heptanucleotide 5-- pGCGCATG. The results obtained indicate that restrictase Hin 1 II cleaves the substrate

S -SATS S;

3 -GTAC-5,

f.

Claims (1)

in the places indicated by arrows. Invention Formula A method of producing endonuclease-restriction enzymes that have the ability to recognize and cleave the sequence of the nucleotides 5 -GPuCGPyC-3 and | b-SATS-3, including cultivating the producer to achieve the desired n iOTHOCTH cells on a liquid nutrient medium containing the brain extract and biosynthetic factors}. for the enzyme, cell destruction by ultrasound, and the subsequent preparation of the target product using chromatographic purification on phosphocellulose in potassium phosphate buffer and elution of sodium chloride gradient enzymes j in order to simplify 0 of the method of obtaining and increasing the yield of the target products, as a producer use the strain Haemophilus influenzae VKPM B-4035, after chromatographic purification of restrictases, 5 separating into phosphocellulose, further purifying for a restriction enzyme that recognizes and cleaves the nucleotide sequence of 5 -GPuCGPyC-3, by chromatography 0 on blue sepharose and heparinepharose with elution of the enzyme with buffer containing 0.2 M NaCl, during chromatography on blue sepharose, and a gradient of NaCl of 0.2-0.6 M during chromatography on 25 heparinsepharose, and chromatographic purification of a restriction enzyme that recognizes and cleaves the nucleotide sequence 5 -CATG 3, is performed on DEAE-cellulose and heparin-sepharose 30 with an enzyme zlation with a NaCl gradient of 0.0-0.5 M and 0.1-1.0 M NaCl, respectively.