HU197348B - Process for producing 5-atgcat-specific restiction endonuklease - Google Patents

Abstract

Specifically restricting 5-ATGCAT endonuclease enzyme is prepd. by fermenting serotype stock 'C' of the streptococcus mutans bacterium. The latter is deposited in the National Collection of Microorganisms under No.00338 at the National Institute of Public Health. Following fermentation the cellular mass which contains the enzyme is separated and rendered accessible. Nutrient consisting of heart-brain extract is suitable for, preferably aerobic fermentation.

Description

This invention relates to a 5-ATGCAT-specific restriction endonuclease (SmucI) from a Streptococcus mutant .c 'serotype.

It is known that most bacterially produced sequence-specific endonucleases, or commonly known as restriction endonucleases, recognize double-stranded nucleotide structures of 4 or 6 nucleotides in double-stranded DNA. Reactions catalyzed by the functional group of these enzymes catalyze the reaction of the PNA cleavage within or directly adjacent to the recognition sequence.

It is also known that the importance of restriction endonucleases lies in this specific recognition and cleavage, which has made it possible to study the inheritance of living organisms of various levels of organization by isolating, analyzing, recombining and altering genes from complex genomes. In addition to the physical mapping of genes, restriction endonucleases also formed the basis of genetic engineering technologies.

Restriction endonuclease production can be detected in more than one hundred bacterial species.

Many known restriction endonucleases have the same recognizable cleavage site. These enzymes, isolated from different species but recognizing the same structure, are referred to as muscle isomers.

The existence of muscle isomers allows the practical, or economic, considerations of optimum enzyme.

Due to the properties of the recognition sequence of the restriction endonuclease Ava III produced by Anabaena variable algae, the so-called Ava III. can be classified into a high specificity group, making it an important tool in genetic engineering research.

The disadvantages of AVA III are:

(a) Variable cultivation of Anabaena is carried out on medium which is rarely used in bacteriology. Cultivation takes 8 to 10 days and requires special illumination and constant flow of carbon dioxide all the time (FEBS Letters 104, 39-44, 1979).

(b) Ava III is not, according to the available data, a pure production and is therefore not engaged in production.

Another recently recognized iso-isoenzyme is the restriction endonuclease Nsil produced by the bacterial strain Neisseria sicca. This enzyme also recognizes and cuts in the same place as the previous one, but the disadvantage of commercially available preparations is both the high price and the instability and rapid inactivation of the enzyme.

SUMMARY OF THE INVENTION It is an object of the present invention to provide a restriction endonuclease with Ava ΙΠ which is readily available and economically obtainable.

The method of producing the 5-ATGCAT-specific restriction endonuclease according to the invention is characterized in that a strain of the .c 'serotype of the bacterium Streptococcus mutant (National Institute of Public Health, National Collection of Microorganisms, 00 338, National Collection of Microorganisms / The enzyme-containing cells from the fermentation broth are isolated and disrupted in a known manner. The culturing is conveniently carried out under aerobic conditions.

According to a preferred embodiment of the invention, a heart-brain extract is used for culture. The bacterium can be cultured at 22 ° C to 40 ° C, preferably 37 ° C. The cells from their resuspension are pelleted by centrifugation, the cells are lysed by sonication, and then purified from the protein solution by ultracentrifugation by gel filtration, hydroxylapatite chromatography, ammonium sulfate precipitation and hydrophobic chromatography, or by enzymatic lysis,

Characteristics of the bacterium referred to:

Microscopic morphology: Most of the diplococcus, 0.6-0.8 micrometer in size, form a long chain in a liquid medium with a slightly elongated flame tongue. Its staining is Gram-positive, and degenerate forms can also be Gram-negative in aging cultures.

Colony morphology: White, round, slowly growing colonies with a diameter of about 1 mm over 2 days in an aerobic culture on an agar plate containing Brain-Heart Infusion (Difco). Liquid in culture: Causes uniform turbidity in 37 ° C aerobically shaken culture.

Maintenance: Weekly passage on inclined agar medium containing heart-brain extract or stored at -20 ° C in 50% glycerol.

Preparative Bacterial Production: The bacterium was cultured in a manner known per se, with or without shaking, in culture media containing various combinations of meat extract, yeast extract and heart-brain extract.

Bacterial growth at 37 ° C is optimal even without continuous agitation and shaking. Cell proliferation can be monitored by measuring optical density at 550 nm. As soon as the culture reaches the end of the logarithmic phase (this heart-brain extract is grown on medium at 0.8-0.9 units as measured in a 10 nm cuvette, mog bouillon-yeast extract at 0.6-0.6 units). ), the culture was cooled in an ice bath and centrifuged at 10,000 g for 30 min at 0 ° C, cell mass was 3 mM TrisHCl (Tris-hydroxymethylaminomethane), pH 8.1 mM EDTA (ethylene diamine). -tetraacetate) and washed with 7 mM 2-mercaptoethanol. This bacterial cell mass can be frozen indefinitely and stored without loss of enzyme at -20 ° C or used immediately to digest the enzyme.

For enzyme digestion, 2 g wet weight cells were suspended in 2 ml buffer containing 20 mM TrisHCl (pH8), 7 mM 2-mercaptoelanol. Cell lysis was performed with an ultrasonic disintegrator in an ice bath. The 10-second pulses were followed by a 1-minute pause, and the digestion was continued until the optical density of the suspension dropped below 10% of the original value. This usually occurred after 20-25 pulses were applied. Thus, sufficient enzyme activity has been released in the suspension. The yield can be increased by addition of Triton X 100 (Fluka, Switzerland) with a nonionic delergent, adjusting the suspension to a final concentration of 0.05% to 2%, preferably 1%. Subsequently, in the alternative method, RNase (Reanal) was added to the suspension to remove RNAs, and was stirred at 200 micrograms / ml and stirred at 0 ° C for 30 minutes. Hereafter phosphocellulose. powder (Whatman P 11), which was activated in the usual manner, was removed for 20 minutes at 0 ° C to remove most of the non-specific nucleases.

Cellular debris, essentially free of enzyme, was removed by ultracentrifugation (100,000 g, 60 minutes). We have found that lower settling speeds are also satisfactory (Janetzky K24 centrifuge, +4 ° C, 14,000 rpm, 60 minutes).

The enzyme was purified from the 2 ml supernatant by gel filtration chromatography in the first step. The column packed with Sephadex G 200 or Sephadex 6 B (Swedish Pharmacia) was chromatographed in a buffer containing 20 mM TrisHCl, pH 8. 7 mM 2-mercaptoethanol and 1 M sodium chloride. Flow was performed with a peristaltic pump at a flow rate of 5 ml / h. The active fractions were pooled and the enzyme was further purified by hydroxylapatite (Bio-Rad, USA) chromatography.

The hydroxylapatite column was washed with a buffer containing 1 mM sodium potassium phosphate (pH 7.4) and 7 mM 2-inertapto-ethanol, and the combined active fractions were then loaded onto the column by the addition of 1 nM phosphate buffer. After loading, the column was eluted with a gradient of 1 mM to 400 mM phosphate containing 7 mM 2-mercaptoethanol and the active fractions pooled.

In the second step of enzyme purification, residual impurity nucleases were precipitated from the combined active fractions with ammonium sulfate. For the enzyme preparation, 4 of a saturated solution of ammonium sulfate, adjusted to pH 8, were slowly stirred in ice to give a 25% (NH *) 2 SO 2 saturated solution and stirred in an ice bath for 60 minutes. The precipitate was then pelleted with a Janetzky K 24 centrifuge (+4 ° C, 14,000 rpm, 30 minutes).

The supernatant was finally chromatographed on a Phenyl-Sepharose column previously treated with 25% saturated ammonium sulfate, eluting with a gradient of ammonium sulfate reducing the enzyme bound on the column.

In the alternative method, DEAE cellulose (Whatman DE52) chromatography was used after hydroxylapatite chromatography. The column was pre-treated with 10 mM TrisHCl (pH 8), 1 mM 2-kidney capto-elanol buffer and washed with the same buffer after allowing the previous active fractions to pass. The enzyme was eluted with a gradient of NaCl from 0 to 1 M.

The active fractions containing the enzyme were combined and dialyzed (and concentrated) against 10 µM TrisHCl (50) containing 50% glycerol and 7 mM 2-mercaploethanol buffer. The resulting enzyme preparation can be stored at -20 ° C.

The enzyme preparation, in the degree of purity it produces, meets the requirements for restriction enzymes used in DNA testing and genetic engineering: i.e., it does not contain any other specific nuclease, non-specific nucleases. and no nucleolidase or phosphatase contamination is detected. The absence of the enzyme composition from specific and non-specific riucleases was demonstrated by the fact that a series of fragments of lambda DNA or adenovirus DNA with the enzyme composition did not show any new fragment upon digestion of the enzyme over 50 hours or digestion of the fragments. suggestive of smear. The absence of exonucleases and nucleotidases is evidenced by the conversion of isotopically labeled native or digested phage PM2 DNA to less than 0.1% of the radioactivity after acid digestion (an indicator of degradation to nucleotides). Phosphatase contamination was excluded by spectrophotometric measurement in the absence of degradation of a chromogenic substrate, nitrophenyl phosphate.

In the assays, the enzyme activity was measured by titration (Roberts et al., J. Mol. Bio. 91, 121-3, 1975). By diluting the enzyme, the smallest amount of enzyme complete with 1 microgram of lambda phage DNA in 60 minutes can be determined. digestive ad, under standard conditions. (25 mM TrisHCl (pH 7.5); 10 nM magnesium chloride; 10 nM 2-mercaptoethanol; 150 mM sodium chloride, 37 ° C)

The recognition specificity of the enzyme was determined by a computerized method. We searched the locations of the various hexanucleotide combinations in the memory of the SV40 viral DNA in the computer memory for probable different data, and compared the resulting computer maps with the real map determined by biochemical, enzymatic methods on the SV40 DNA molecule. thus, the enzyme cleavage recognition sites could be localized in sequences where the computer recognized the 5-ATGCAT hexanucleotide.

We also determined the exact location of the intersection within the recognition site. Terminal fragments of type 1 adenovirus DNA cut with restriction enzyme PstI were ligated with T4 ligase to terminal fragments of adenoviral DNA cleaved with the enzyme composition of the invention. The two types of fragments could be ligated, which was only possible if the enzyme of the invention and the restriction endonuclease PstI produced complementary sticky ends of the same structure. (Symmetrically split double-stranded .blunt end 'ligation is excluded). Given that the inner four nucleotides in the recognition sequence of the PstI enzyme are identical to the inner four nucleotides of the restriction endonuclease recognition sequence we isolated, and that the fragments produced by the PstI endonuclease are known to have 3 'overhangs (extension)

PstI: 5-CTGCAG 3 '

Str. Mutant .c I enzyme (SniucI): 5-ATGCAT 3 ', thus the ligatability proves that the enzyme (SmucI) of the invention also cleaves 3' overhanging single-strand ends. Thus, the exact location of the intersection can be located between the fifth adenine and the sixth limb of the recognition sequence:

5-ATGCAT 3 '

The main advantages of the process according to the invention are as follows:

a) The enzyme content of the deposited Streptococcus mutant serotype .c isolate is extremely high.

b) The enzyme is very stable during the process. After any purification step, the product can be stored for weeks at + 4 ° C with minimal losses. Other types of enzymes are completely degraded during this time. (After 1 month, 70-80% of the original activity remains.)

c) Due to the high enzyme content of the bacterium and the small amount of readily separable contaminating nuclease, the process utilizes few simple purification steps to produce a high activity, high purity enzyme composition.

d) The process results in an enzyme product having a new recognizing specificity. It is not available due to the complex production and difficult to purify of the Ava III isomer.

On the other hand, with respect to the other known muscle isomer, Nsil, the less expensive preparation and greater stability of the enzyme according to the invention are advantageous.

e) Due to the properties of the recognition sequence of the enzyme according to the invention, it can be classified into the high specificity group, thus cutting pieces of DNA corresponding to the average size of a gene. These fragments can be inserted and ligated at the PstI cleavage site of the commonly used cloning vector plasmid, pBR322.

The process of the invention is illustrated by the following embodiment:

Example 1

50% glycerol suspension of Streptococcus mutant serotype c at -20 ° C was inoculated with 50 ml of cardiac brain extract medium (prepared and sterilized according to Difeo, USA). After shaking for 24 hours at 37 ° C, the whole was inoculated into a 2-liter heart-brain extract medium in a 3-L Erlenmeyer flask. The culture was aerobically cultured at 37 ° C without shaking and the optical density measured at 550 rin was checked. The culture reached the end of the logarithmic phase of growth at 0.9 units, then cooled in an ice bath, and the bacterial cells were pelleted at 10,000 g for 20 minutes at +4 ° C and 10 mM TrisHCl, pH 8; 7 mM 2-inercaptoethanol; Washed with 1 mM EDTA buffer. The resulting approx. 2 g of bacterial cell mass were suspended in 2 ml of 20 µM TrisHCl (pH 8), 7 mM 2-mercaptoethanol buffer, followed by 25 min total incubation with an MSE ultrasonic device in an ice bath, with one-minute cooling intervals. The suspension was then mixed with Triton XI00 detergent to a final concentration of 1%.

The digested slurry was then pelleted by Janetzky Vac 602 ultracentrifuge at 30,000 rpm for 60 minutes (100,000 g) at +4 ° C. The 2 ml supernatant was further purified by gel filtration.

A 1.5 cin packed with ² x 80 cm Sephadex 0200 (Pharmacia) gel particles and 1 M NaCl; 10 mM TrisHCl pH 8.7, saturated with 7 mM 2-mercaptoethanol buffer. the supernatant was eluted at a flow rate of 5 ml / h. One ml fractions were collected, enzyme activities were tested, the active fractions pooled, and the enzyme was bound to a hydroxylapatite column (1.5 cm ² x 5 cm) after addition of 1 mM phosphate buffer (pH 7.4). buffer, containing 1 mM phosphate and 7 mM 2-mercaplooethanol at a flow rate of 5 ml / h. Subsequently, the column was decontaminated with a total of 20 mL of phosphate gradient from 1 mM to 400 mM phosphate. The enzyme is 5

Between 100 mM and 180 mM phosphate was dissolved from the column. The active fractions were combined and then saturated (pH 8) ammonium sulfate (25 mL) was added slowly with ice-cooling to a pH of 25% (NhhSCh) and stirred overnight in an ice bath. the precipitate was centrifuged in a Janetzky K 24 centrifuge at +4 ° C and 14,000 rpm for 30 minutes and the supernatant was finally purified by hydrophobic chromatography on a Phenyl Sepharose (Pharmacia) column (1.5 cin ² x 15 cm) previously 25% ammonium 10 nM TrisHCl (pH 8);

Wash with mM 2-mercaptoethanol solution. The unbound material was also washed with this buffer. The enzyme was eluted from the column with a gradient of 25% to 0% ammonium sulfate in buffer containing 10 mM TrisHCl (pH 8) mM 2-mercaptoethanol.

After combining the active fractions, dialysis and concentration was performed with 50% glycerol;

mM TrisHCl (pH 8) against 7 mM 2-mercaptoethanol solution at + 4 ° C for 12 hours. The enzyme preparation can thus be stored at -20 ° C.

The yield is about 60% (20,000 units of enzyme activity per 2 g cells).

Example 2

50 ml of a suspension of 50% glycerol in a serolipid strain of Streptococcus mutant c was seeded with 50 ml of cardiac brain extract medium. After culturing at 37 ° C for 24 hours, the whole was inoculated into a 2 liter medium in a three-liter Erlenmeyer flask containing the following: 40 1.1% Meat Extracts (Bouillon Barley, Human, Hungary); 1% yeast extract (Bacto Yeast Extrase, Difco, USA). The pH of the medium was adjusted to 7.6 and sterilized by autoclaving. The culture reached the end of the logarithmic phase at about 0.5 550 nm at 45 550 nm in this medium. At this time, the culture was chilled in ice and proceeded as in Example 1 below. 50

Example 3

The bacterial production and cell mass 55 were sonicated as in Example 1. However, after Triton treatment, 200 micrograms / ml RNase was added to the suspension and kept in an ice bath for 30 minutes, then mixed with 0.5 g of activated phosphocellulose powder.

Subsequently, ultraceritrifugation, Sephadex 0200 and hydroxylupulin chromatography were performed as described in Example 1.

The final step was the use of a DEAE cellulose column gasket. Active fractions of the previous hydroxylapatite chromatography were passed through a DEAE cellulose column (1.5m ² x 5 cm) previously washed with 10mM TrisHCl (pH 8), 7mM 2-mercapto-enol buffer at 5 ml / h. After washing the column with the above buffer, the enzyme was eluted with 40 mL of sodium chloride buffer, rising from 0 to 1 M. The activity was detected in fractions between 0.4 M and 0.8 M.

Combining, dialyzing and storing the active fractions is a. Example 1.

Claims (6)

PATENT CLAIMS A method for the production of a 5 'ATGCAT-specific restriction endonuclease by fermentation comprising culturing a strain of the. the cell mass is isolated and digested in a known manner and then purified by its enzyme. (Priority: January 16, 1986) The method for cultivating the strain according to claim 1, wherein the culturing is carried out under aerobic conditions. (Priority: 16 Jan 198G) The method for cultivating the strain according to claim 1, wherein the medium is heart-brain extract. (Priority: January 16, 1986) The method for cultivating a strain according to claim 1, wherein the medium is yeast and meat extract medium. (Priority: January 16, 1986) The method for purifying the enzyme according to claim 1, wherein the bacterial cells are purified from the bacterial cells by the addition of Triton X100 after sonication. (Priority: January 16, 1986) 6. Method for purifying the enzyme according to any one of claims 1 to 5, characterized in that the digested enzyme is treated with RNase, the crude enzyme deprotected with RNA being treated with phosphocellulose and DEAE cellulose ion exchange chromatography to remove contaminating nucleases. (Priority: September 9, 1987)