HU204082B - Process for producing ceqi restriction enzyme - Google Patents

Abstract

A ceql restrictive enzyme contg. a GATAC recognition and a GAT-ATC cleavage point, is prepd. by growing corynebacterium equiphylum (registered under OK100355) or mutants or variants of above producing ceql enzyme, on substrates contg. accessible carbon, nitrogen and mineral salt nutrients. The separated enzyme can be optionally purified. (Corrected abstract)

Description

The present invention relates to a novel Ceq1 restriction enzyme.

Restriction endonuclease enzymes are proteins that can cleave DNA (the DNA of the clock). It is a means of defense that is widespread among bacteria: it kills the germs of viruses (phages) that penetrate into the cell.

Their significance is that the long chain of DNA is not cleaved, but in clearly defined sites (s); this feature makes them suitable for the purposes of science known as "recombinant DNA technology" or more commonly known as genetic engineering.

DNA is an extremely long linear polymer containing four kinds of nulldeotides in which the molecule is made up of two mutually determining chains with opposite chemical directions. The "meaning" of DNA is given by the order of the bases, so the base order is not statistic, but according to the laws of large numbers, virtually every combination can be found in it, with only a different frequency.

By artificially combining naturally occurring (bacterial, mammalian, viral, etc.) genes and DNA fragments (oligonucleotides), it is possible to eddignem existing new properties, new properties of organisms.

This work requires restriction endonucleases which cut the DNA at the desired site (s) (The pieces can then be assembled in the desired order using another enzyme, DNA ligase).

The number of restriction enzymes described to date is close to a400, and once a year the full list of R. J. Roberts is published. (Recent: NucleicAcidResearch (1984), 12x167).

The enzymes recognize a sequence of 4,5,6 or 8 bases and are usually cleaved within this sequence. While single sequences can be cleaved by a large number of enzymes from a variety of bacteria, there are many sequences for which no enzyme has been found.

The more complete the toolbox, the more successful genetic engineering is, and thus the interest in new cleavage enzymes is understandable. (The recognition site and the cleavage itefy are not the same, because the same 6-base sequence, i.e. the recognition site, can be cleaved in several ways, and even some of the enzyme cleavage sites are farther away from the first recognition site.)

The bacterial strain we isolate produces a restriction enzyme (Ceql). which recognizes 5 GATAIC 3 sequences and cleaves it to produce a GAT and an ATC terminus. For the identification of recognition and cleavage sites, extensive proof work has been carried out with a lack of direct sequence data.Roberts cited another of the two enzymes that recognize the GATATC sequence and cleave in the same way; this is the EcoRV. Thus, our enzyme can be considered as the Eco Ris isomer.

The identity of the cleavage of Ceql and EcoRV is described in la, ib and le. illustrates:

The la. Figure 1A shows that the fragments created by the two enzymes are the same size. Lambda phage DNA (sample A) was cleaved with EcoRVenzyme φ sample), EcoRV enzyme and Ceql enzyme (sample c) and Ceql enzyme (sample d).

The lb. Fig. 4A shows the localization of the Ceq1 enzyme on the DNA of plasmid pBR322. Fusion of DNA fragments double digested with enzymes defined the enzyme cleavage site.

That's down. Fig. 4A shows the sequencing strategy and sequencing result of the Ceq1 enzyme cleavage site.

Figure 2 shows purification of Ceql enzyme by DEAE cellulose chromatography The column shows the enzyme activity of the fractions eluted from the column, the arrow indicating the appearance of a buffer at 200 mM. The strain is intended to be a coincidental isolate. Its striking color is intense. OKI and Szegedi Kálal are defined as Coryne bacteria and are probably identical to Corynebacterium equii.

It is not picky in media solutions, any so-called.

in complete medium (e.g. 10 g protein hydrolyzate, 5 g yeast extract, 10 g NaCl per liter), and the temperature is not critical *, does not grow much better at about 37 degrees than about 4 degrees. Acolonia are very variable in size, dense and tend to be very tiny30.

The strain was deposited at the National Institute of Public Health on April 23, 1986, under OKI 00355. The strain is maintained by the National Collection of Agricultural and Industrial Microorganisms (NCA35 IM), my number 000533

According to the present invention, the Corynebacterium equii strain OKI00355 or a Ceql enzyme-producing mutant or variant thereof is cultivated on nutrient soil containing assimilable carbon and nitrogen sources and mineral salts, and the resulting enzyme is isolated and purified if desired.

The enzyme is purified from late hanging or stationary phase, i.e., old cells, which contain many enzymes. There are many ways to destroy cells: ultrasound, Frenchpress, rubbing, and even blending with a mixture of carbonated snow and glass beads in a coffee grinder.

The composition of the digestion solution is non-critical, at pH-7, not too low an ionic-strength puff forces

After digestion, the bacterial debris is removed by centrifugation and the enzyme is precipitated from the supernatant with 70% saturated ammonium sulfate. (It is preferable to precipitate the remaining nucleic acids prior to precipitation by adding 0.1 to 55 volumes of 10% streptomycin sulfate and centrifuging the precipitate.)

The enzyme precipitated with ammonium sulfate can be purified from salts by dialysis or gel filtration (dialyzed overnight against 100 volumes of 10mMpH-7 phosphate buffer 50) and then purified by ion exchange chromatography2

HU 204082 Β continue to work.

The following materials are suitable for this purpose: DEAE cellulose (or DEAE-Sephadex, Sephacell, etc.), aminopentyl or aminohexyl agaroses, MonoQ PPLC column.

The enzyme does not bind to CM and phosphocellulose, Heparin or Blue agarose. It is eluted from the ADEAE column at a salt concentration of about 200 mM KCl (gradient), whereas it is capable of dissolving 400 mM KCl from aminopentyl agarose and MonoQ. In each case, the buffer may be phosphate or tris-HCl at about pH 7.

The enzyme can then be dialyzed against 50% glycerol and stored at -20 ° C without loss of activity. The preparation is far from being biochemically pure, but is excellent for genetic engineering: it does not contain any other endonuclease, no exonucleases at the ends of the DNA chain, or no phosphatases to remove the phosphate at the end of the DNA - no enzyme contamination it would cause unwanted transformation of the DNA it digests.

The enzyme has the advantage that it has a high specificity (a 6 base sequence is less common than a 4 base recognition site), is relatively high in amount and can be easily purified. The latter is particularly important because the purification of the isomeric isomer at our Institute is very difficult. Another great advantage of our enzyme is that it is extremely stable and does not lose activity at room temperature, which, for example, greatly simplifies its transport.

The process of the invention is illustrated by the following example.

Example

The bacterium, previously defined as Corynebacterium equii, in solid medium (petri dish, complete nutrient: 10 g protein hydrolyzate, 5 g yeast extract, 10 g NaCl and 15 g per 1000 ml) was grown at 37 degrees and air-tight for 1 week (until use). Store in a sealed polyethylene bag at 4 ° C.

The intense color of the bacterial mass is called so called. Scrape with a "rubber pliceman" and sonicate in 10 volumes (about 2 ml) of 10 mM Tris-HCl, pH 7.4 in 50 mM NaCl. (B-30 Branson sonifier, 2 min, 2.5 output, strong cooling applied.)

The digested suspension was centrifuged in an Eppendorf table top centrifuge (max speed) for 20 minutes in a cold room and the supernatant was mixed with 0.2 ml of 10% streptomycin sulfate solution in ice. After half an hour, the suspension was centrifuged again (5 minutes, Eppendorf cf. In a cold room), and the resulting supernatant was added with a solid, neutral ammonium sulfate solution to 70% saturation. (To the acidic ammonium sulfate is added 0.1 milligrams of tris base, which results in a neutral chemistry for the Merck ammonium sulfate and Sigma tris used. 70% saturation corresponds to 470 g / l solid ammonium sulfate.)

One hour later, the precipitate containing the enzyme can be removed from the cold mixture by further centrifugation (above 1000 g) for 5 minutes.

Tris-HCl, dissolved in pH 7.4 buffer, and then dialyzed against the same buffer overnight to prepare a "crude extract," which is suitable for DNA analytical, mapping, but not purely genetic engineering.

Further purification of the crude extract was performed on a DEAE cellulose column. Whatman DE52 cellulose (Whatman, Sprmgfiels Mill, Maidstone, Kent, England) 2 x 5 cm column was equilibrated with 10 mM, pH 7.4 tris-HCl, 1 mM EDTA, 10 mM mercaptoethanol and washed after application of the sample. The column is then washed with 150 and 200 mM KCl solutions in the above buffer. The enzyme is eluted with 200 mM solution and has a purity suitable for genetic engineering purposes: the digested DNA fragments remain the same size in the case of 100-fold digestion and can be ligated with DNA ligase. The enzyme can be re-cleaved with the enzyme (up to 8090%), amounting to 8000 International Units, and losing less than 10% of its activity in one year at -20 ° C.

The definition of an enzyme unit is the amount of enzyme molecule, according to the internationally accepted unit, that completely digests one µg of DNA per hour under optimal conditions in 20 µl of reaction mixture.

Optimal conditions for cleavage with the above enzyme: 150 mM NaCl or KCl, 5-10 mM Mga ₂ , 10-50 mM tris [tris (hydroxymethylamino) methane] HCl buffer, pH 8.0, optimum temperature: 37 -42'C.

The recognition and cleavage sites were identified as follows:

During double digestion of pBR322 with the new enzyme and EcoRI, PvuH, HindIII and BamHI, the cleavage site was located between 180-195 bp, with only one palindrome sequence: the GATATC sequence of EcoRV. This is illustrated in lb. figure.

The Ceql cleavage site was verified by sequencing. Plasmid pBR322 was digested with BstN1 (Biolalas, New England) and the 130-1059 bp C fragment was isolated, labeled with ^3z P) dATP (by Maniatis) and then BamHI (manufactured by SZBK) using Klenow polymerase (BoelvingerManuheim). cleavage resulted in a 245 bp fragment (130-375 bp). The fragment was sequenced and run on the same gel with Ceq1-cleaved fragments (see Figure lanes 1-5).

Plasmid pKL300 was digested with Hinf (Biolalas) and fragment (18-785) C was isolated after the end-labeled sequence, and the AHinf-SalI fragment (514-785 bp) was sequenced and run with the Hinf-Ceq1 fragment. (See Figure 6, lanes 6-10). The above operations were performed according to the manufacturer's specifications. Plasmid pKL300 is described by McKenney, K. 1981: Gene Amplification and Analysis, Vol H, 383-386.

Cleavage of the synthetic oligonucleotides confirmed that digestion of GGGATATTCCC and CCCGATATCGGG oligomers resulted in two 5 and 6 digestions, respectively.

A product consisting of HU 204082 űk nucleotides was generated (cleavage site underlined).

Claims (2)

A process for the preparation of a Ceql restriction enzyme having a GAIATC recognition site and a GATATC cleavage site, wherein the Ceynehaeterium equii strain NCAIM 000533 or a Ceql enzyme-producing mutant or variant thereof is assimilated with a carbonate and nitrogen source and mineralized. 2. The method of claim 1, wherein the culture is carried out on a solid medium