WO1991013975A1 - CLONAGE ET EXPRESSION D'ENDONUCLEASES DE RESTRICTION DE $i(NEISSERIA) - Google Patents

CLONAGE ET EXPRESSION D'ENDONUCLEASES DE RESTRICTION DE $i(NEISSERIA) Download PDF

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WO1991013975A1
WO1991013975A1 PCT/US1991/001599 US9101599W WO9113975A1 WO 1991013975 A1 WO1991013975 A1 WO 1991013975A1 US 9101599 W US9101599 W US 9101599W WO 9113975 A1 WO9113975 A1 WO 9113975A1
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restriction endonuclease
ngoaiv
dna
recombinant
host
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PCT/US1991/001599
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Alan Wayne Hammond
Deb Kumar Chatterjee
Gary Floyd Gerard
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Life Technologies, Inc.
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1003—Transferases (2.) transferring one-carbon groups (2.1)
    • C12N9/1007—Methyltransferases (general) (2.1.1.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses

Definitions

  • This invention is directed to a restriction endonuclease and modification methylase from Neisseria gonorrhoeae called NgoAIV and M ⁇ NqoAIV, respectively.
  • This invention is further directed to isoschizomers of NgoAIV which cleave the same phosphodiester bonds within the same recognition seguence as NgoAIV, and isoschizomers of M ⁇ NgoAIV that chemically modify the same nucleotides within the same recognition sequence as M ⁇ NgoAIV.
  • Restriction endonucleases are a class of enzymes that occur naturally in prokaryotic and eukaryotic organisms. When they are purified away from other contaminating cellular components, restriction endonucleases can be used in the laboratory to cleave DNA molecules into precise fragments. This property enables DNA molecules to be uniquely identified and to be fractionated into their constituent genes. Restriction endonucleases have proved to be indispensable tools in modern genetic research. They are the biochemical "scissors" by means of which genetic engineering and analysis are performed.
  • Restriction endonucleases act by recognizing and binding to particular seguences of nucleotides (the "recognition sequence") along the DNA molecule. Once bound, they cleave the molecule within, or to one side of, this sequence.
  • Different restriction endonucleases have affinity for different recognition sequences. About 100 kinds of different endonucleases have so far been isolated from many microorganisms, each being identified by the specific base sequence it recognizes and by the cleavage pattern it exhibits.
  • restriction endonucleases called restriction endonuclease isoschizomers, have been isolated from different microorganisms which in fact recognize the same recognition sequence as those restriction endonucleases that have previously been identified. These isoschizomers, however, may or may not cleave same phosphodiester bond as the previously identified restriction endonuclease.
  • restriction endonucleases play a protective role in the welfare of the microbial cell. They enable the microorganism to resist infection by foreign DNA molecules like viruses and plasmids that would otherwise destroy or parasitize them. They achieve this resistance by scanning the lengths of the infecting DNA molecule and cleaving them each time that the recognition sequence occurs. The DNA cleavage that takes place disables many of the infecting genes and renders the DNA susceptible to further degradation by non-specific exonucleases.
  • a second component of microbial protective systems are the modification methylases.
  • Modification methylases are complementary to their corresponding restriction endonucleases in that they recognize and bind to the same recognition sequence.
  • Modification methylases in contrast to restriction endonucleases, chemically modify certain nucleotides within the recognition sequence by addition of a methyl group. Following this methylation, the recognition sequence is no longer bound or cleaved by the restriction endonuclease.
  • the microbial cell modifies its DNA by virtue of its modification methylases and therefore is completely insensitive to the presence of its endogenous restriction endonucleases.
  • endogenous restriction endonuclease and modification methylase provide the means by which a microorganism is able to identify and protect its own DNA, while destroying unmodified foreign DNA.
  • restriction-modification system The combined activities of the restriction endonuclease and the modification methylase are referred to as the restriction-modification system.
  • Three types of restriction-modification systems have been identified that differ according to their subunit structure, substrate requirements and DNA cleavage. Specifically, Type-I and Type-Ill restriction systems carry both modification and ATP-requiring restriction (cleavage) activity in the same protein.
  • Type-II restriction-modification systems consist of a separate restriction endonuclease and modification methylase, i.e., the two activities are associated with independent proteins.
  • Type II restriction endonucleases are endodeoxy- ribonucleases which are commonly used in modern genetic research. These enzymes recognize and bind to particular DNA sequences and once bound, cleave within or near this recognition seguence. Phosphodiester bonds are thereby hydrolyzed in the double stranded DNA target sequence, i.e., one in each polynucleotide strand.
  • Type-II restriction endonucleases can generate staggered breaks within or near the DNA recognition seguence to produce fragments of DNA with 5' protruding termini, or DNA fragments with 3' protruding termini.
  • Other Type-II restriction endonucleases which cleave at the axis of symmetry, produce blunt ended DNA fragments. Therefore, Type-II restriction endonucleases can differ according to their recognition sequence and/or the location of cleavage within that recognition sequence.
  • Type-II restriction endonucleases are frequently used by the genetic engineers to manipulate DNA in order to create novel recombinant molecules. Specific Type-II restriction endonucleases are known for numerous DNA sequences, but there is still a need to provide new Type-II restriction endonucleases. These new enzymes will add to the list of indispensable tools needed for modern genetic research.
  • the present invention is directed to a new Type II restriction endonuclease, NgoAIV, with a process for obtaining it and with the use thereof.
  • restriction endonuclease is called NgoAIV and its modification methylase is called M ⁇ NgoAIV.
  • This invention is also directed to a method for producing a DNA fragment comprising contacting a sample of DNA with a restriction endonuclease which recognizes the palindromic sequence:
  • Figure 1 shows a simplified restriction map of plasmid DNA, pRMNgoAIV. This plasmid contains and expresses the genes encoding NgoAIV and M ⁇ NgoAIV. Definitions
  • rDNA recombinant DNA
  • Cloning vector A plasmid or phage DNA or other DNA sequence which is able to replicate autonomously in a host cell, and which is characterized by one or a small number of endonuclease recognition sites at which such DNA sequences may be cut in a determinable fashion without loss of an essential biological function of the vector, and into which DNA may be spliced in order to bring about its replication and cloning.
  • the cloning vector may further contain a marker suitable for use in the identification of cells transformed with the cloning vector. Markers, for example, are tetracycline resistance or ampicillin resistance.
  • Expression vector A vector similar to a cloning vector but which is capable of enhancing the expression of a gene which has been cloned into it, after transformation into a host.
  • the cloned gene is usually placed under the control of (i.e., operably linked to) certain control sequences such as promoter sequences.
  • Restriction endonuclease isoschizomer A restriction endonuclease isoschizomer is a term used to designate a group of restriction endonucleases that recognize and bind to the same recognition sequence but are isolated from different microbial sources. Restriction endonucleases isoschizomers may or may not cleave in the exact location as the restriction endonuclease with which it is being compared. Modification methylase isoschizomer. A modification methylase isoschizomer is a term used to designate a group of modification methylases that recognize the same recognition sequence but are isolated from different microbial sources. Modification methylase isoschizomers may or may not chemically modify the same nucleotides within the recognition sequence as the modification methylase with which it is being compared.
  • Recognition sequences are particular sequences which restriction endonucleases and modification methylases recognize and bind along the DNA molecule. Recognition sequences are typically four to six (and in some cases eight) nucleotides in length with a two fold axis of symmetry.
  • Recombinant host Any prokaryotic or eukaryotic organism which contains the desired cloned genes on an expression vector or cloning vector.
  • Promoter A DNA sequence generally described as the 5' region of a gene, located proximal to the start codon. At the promoter region, transcription of an adjacent gene(s) is initiated.
  • Structural gene A DNA sequence that is transcribed into messenger RNA that is then translated into a sequence of amino acids characteristic of a specific polypeptide.
  • Operably linked means that the promoter controls the initiation of the expression of the polypeptide encoded by the structural gene.
  • Expression is the process by which a structural gene produces a polypeptide. It involves transcription of the gene into messenger RNA (mRNA) and the translation of such mRNA into polypeptide(s) Substantially pure.
  • mRNA messenger RNA
  • the desired purified enzyme is essentially free from contaminating cellular components, said components being associated with the desired enzyme in nature. Contaminating cellular components may include, but are not limited to, phosphatases, exonucleases or undesirable endonucleases.
  • DNA molecule As used herein means double stranded DNA cloning or expression vectors, or DNA sequences which are intergrated into the host genome.
  • NgoAIV recognizes the palindromic seguence 5' GCCGGC 3' and cleaves between the first G and C residues from the 5' end, producing a four-base 5' extension.
  • This invention is also directed to the modification methylase, M ⁇ NgoAIV.
  • M ⁇ NgoAIV recognizes and binds to the same recognition sequence as NgoAIV.
  • M ⁇ NgoAIV chemically modifies certain nucleotides within the recognition seguence by addition of a methyl group, thus making the modified sequence resistant to cleavage with its corresponding restriction endonuclease, NgoAIV.
  • This invention is also concerned with restriction endonuclease isoschizomers of NgoAIV. Specifically, this invention is related to restriction endonuclease isoschizomers of NgoAIV which cleave in the exact location as NgoAIV. Thus, this invention is directed to restriction endonucleases of microorganisms other than Neisseria gonorrhoeae which recognize the recognition sequence 5' GCCGGC 3' and cleaves between the first G and C residues from the 5' end, producing a 4- base 5' extension.
  • This invention is also directed to the modification methylase isoschizomers of M ⁇ NgoAIV.
  • the restriction endonuclease NgoAIV and its modification methylase can be derived from Neisseria gonorrhoeae strain FA1090.
  • the genus Neisseria are Gram negative cocci occurring in pairs or in masses and are aerobic or facultatively anaerobic. These organisms may be found in the oropharynx or nasopharynx and the genitourinary tract of humans and animals.
  • Isoschizomers of these enzymes can be obtained from any genus including, but not limited to, Arthrobacter, Bacillus, Citrobacter, Enterobacter. Escherichia, Flavobacterium, Klebsiella, Micrococcus, Neisseria, Nocardia, Pseudomonas, Salmonella, and Streptomyces.
  • Neisseria meningitidis Neisseria sicca. Neisseria lactamicus, Neisseria mucosa. Neisseria lactamica. Neisseria ovis, Neisseria subflava. and Neisseria flavescens are possible sources of genes expressing isoschizomers of NgoAIV or for isolation of these enzymes.
  • Neisseria might also be used to isolate a modification methylase isoschizomer of M ⁇ NgoAIV or the genes coding for these methylases.
  • NgoAIV and M ⁇ NgoAIV or the genes coding for these enzymes is Neisseria gonorrhoeae as described in the examples.
  • restriction endonucleases or the modification methylases of this invention may be produced by fermentation of the genus Neisseria. Any nutrients that can be assimilated by these strains producing NgoAIV or isoschizomers thereof may be added to the culture medium. Glucose, sucrose, maltose, lactose, glycerol, ethanol, lactates, various fats and oils, and others may be used as carbon source, while yeast extract, peptone, defatted soybeans, corn steep liquor, bouillon and others are suitable as nitrogen source. Minerals and metal salts, e.g., phosphates, potassium salts and magnesium salts, iron, as well as vitamins and growth-promoting substances, may also be added as required.
  • Any nutrients that can be assimilated by these strains producing NgoAIV or isoschizomers thereof may be added to the culture medium.
  • NgoAIV and M ⁇ NgoAIV or their isoschizomers will vary depending on culture conditions. Best results are generally obtained at a temperature of 37°C and at a pH in the range from 3 to 8 under 5% CO 2 conditions; the highest output is achieved in one day by culture with aeration and agitation.
  • One of skill in the art can select the optimal culture conditions for a particular strain on a case by case basis according to the strain used and the composition of the culture medium. Restriction endonucleases produced by the process of this invention are accumulated inside the microbial cells.
  • the microbial cells of the genus Neisseria producing the restriction endonuclease and modification methylase of this invention can be separated from the culture liquid, for example, by centrifugation. Both of these enzymes can be extracted and purified by using known protein purification techniques commonly employed for these types of enzymes.
  • the collected microbial cells are dispersed in a suitable buffer, and then broken down by ultrasonic treatment to allow extraction of the enzyme by the buffer solution. After removal of the residue by ultracentrifugation, ammonium sulfate can be added to the supernatant of the crude lysate for salting out, and the precipitate which separates out is dissolved in a Tris-HCl buffer (pH: 7.6) and dialyzed against a buffer of the same composition.
  • the dialyzed sample can be purified by ion-exchange chromatography, molecular-sieve chromatography and affinity chromatography, giving the restriction endonuclease or modification methylase of this invention.
  • NgoAIV from Neisseria gonorrhoeae
  • the crude lysate is adsorbed directly onto a Affigel blue agarose (Bio-rad Laboratories) column, followed by elution with 0 to 1.5 M potassium chloride solutions.
  • the active fractions collected are then absorbed again on blue agarose and eluted with 0 to 1.5 M potassium chloride solutions.
  • the active fractions are further purified by adsorption on a Mono-Q column and eluted using a gradient of 0 to 1.0 M potassium chloride, affording a standard sample of NgoAIV.
  • assay to detect the presence of the restriction endonucleases and modification methylases can be used during the conventional biochemical purification methods to determine the presence of these enzymes.
  • Restriction endonuclease can be identified on the basis of the cleavage of its recognition seguence.
  • substrate there can be used, for example, Adenovirus- 2 (Ad-2) DNA.
  • Ad-2 Adenovirus- 2
  • the DNA fragments obtained are separated electrophoretically in agarose gels in the buffer systems conventional for the fragment separation in the presence of ethidium bromide.
  • Demonstration of modification methylase activity can be, but is not limited to, a two-step identification process.
  • DNA substrate that contains the recognition sequence (Ad-2 DNA) is incubated with column fractions to be tested for methylase activity.
  • this DNA is then challenged with the corresponding restriction activity to identify those fractions which contain methylase activity. For example, while assaying for M ⁇ NgoAIV, the DNA samples will be challenged with NgoAIV. Thus, DNA samples which do not exhibit cleavage with NgoAIV contain M ⁇ NgoAIV activity.
  • NgoAIV and M ⁇ NgoAIV are preferably obtained by isolating the genes coding for the enzymes and then cloning and expressing them. It is understood in this invention that genes encoding for isoschizomers of NgoAIV and/or modification methylase isoschizomers of M ⁇ NgoAIV can be obtained from microorganisms other than Neisseria gonorrhoeae by using the recombinant technigues described herein. Microorganisms which differ in both genus and species can be used for the purpose of obtaining and expressing the isoschizomers of NgoAIV and M ⁇ NgoAIV.
  • DNA molecules which encode for NgoAIV and/or M ⁇ NgoAIV can be recombined into a cloning vector and introduced into a host cell to enable the expression of the restriction endonuclease or modification methylase by that cell.
  • DNA molecules may be recombined with vector DNA in accordance with conventional technigues, including blunt-ended or stagger-ended termini for ligation, restriction receptor molecule digestion to provide appropriate termini, filling in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and ligation with appropriate ligases.
  • prokaryotic hosts include bacteria such as Escherichia coli, Bacillus, Streptomyces, Pseudomonas, Salmonella, Serratia, Neisseria etc.
  • the most preferred prokaryotic host is E. coli.
  • E. coli has several mechanisms (restriction systems) for identifying foreign DNA and destroying it. This can be a significant problem in a cloning experiment, resulting in reduced recovery of the desired sequences.
  • E. coli contains restriction systems that degrade DNA when it is methylated, either cytosine residues or adenine residues.
  • the well known methylcytosine-specific systems include mcrA (rglA), and mcrB (rglB).
  • the methyladenine-specific restriction system has been designated mrr.
  • the preferred host for cloning and expressing NgoAIV and M ⁇ NgoAIV is an E. coli host in which these restriction systems have been inactivated through mutation or loss.
  • Bacterial hosts of particular interest in the present invention include E. coli K12 strain K802 (mcrA, mcrB, r k - and m k -) and E. coli K12 DH5 ⁇ MCR (F-' endoA1, hsdR17[r k -, m k + ], supE44, thi-1, ⁇ -, recA1, gyrA96, relA1, ⁇ 80dlacZ ⁇ MI5, ⁇ mrr, ⁇ mcrB, mcrA).
  • the prokaryotic host must be compatible with the replicon and control sequences in the cloning vector.
  • NgoAIV and its modification methylase are preferably obtained by isolating the genes coding for the enzymes and then cloning and expressing them.
  • Wilson "Cloned restriction-modification system—a review,” Gene 74:281-289 (1988), describes four techniques for isolating and cloning restriction endonuclease and modification methylases. The four methods reviewed include (1) subcloning of natural plasmids; (2) selection based on phage restriction; (3) selection based on vector modification involving methylation protection; and (4) multi-step isolation. Any one of these four methods can be used for isolating and cloning NgoAIV and M ⁇ NgoAIV.
  • the preferred method according to this invention is vector modification technique, i.e., methylation protection.
  • Methylation protection involves digestion of a plasmid library with the restriction enzyme to be cloned so that only plasmids whose seguences are modified, because of the presence of the methylase, will produce transformants in a suitable host. This selection has worked well to clone endonuclease and methylase genes together as well as methylase genes alone (Szomolanyi et al., 1980; Janulaitis et al., 1982; Walder et al., 1983; Kiss and Baldanf, 1983; and Wilson, 1988).
  • selection based on modification requires that the vector used to construct the plasmid library contain at least one recognition site (recognition sequence) corresponding to the modification methylase to be cloned.
  • Clones that contain the modification gene on the plasmid vector will methylate their own plasmid DNA, provided that the modification methylase is expressed in the host used.
  • plasmid DNA isolated from such clones will therefore be resistant to digestion in vitro by the corresponding restriction endonuclease.
  • Restriction genes and their corresponding modification genes are usually closely linked in the DNA of many bacteria. This being the case, selection for methylase-containing cells can be used as a simple and reliable method for selectively co-isolating methylase and endonuclease clones. In brief, selection of methylase-carrying clones from plasmid libraries which also contain DNA fragments coding for the correspond ing restriction genes frequently results in the isolation of clones that carry both the modification methylase gene and the corresponding restriction endonuclease gene. Methylase-selection is therefore an indirect way of selecting a restriction endonuclease clone.
  • the DNA of the bacterial species to be cloned is purified.
  • the DNA is digested partially with a convenient restriction endonuclease.
  • the resulting fragments are ligated into a cloning vector, such as pCP13 (Darzins, A. et al., J.
  • the DNA/cell mixture is plated on antibiotic media selective for transformed cells. After incubation, the transformed cell colonies are pooled and an aliquot of this cell suspension is grown to create the cell library.
  • the recombinant plasmids are purified in toto from the cell library to make a plasmid library.
  • the plasmid library is then digested to completion in vitro with the restriction enzyme whose corresponding methylase gene is sought. Exonuclease and/or phosphatase may also be added to the digestion to enhance the destruction of non-methylase plasmids.
  • the digested plasmid DNA is transformed into E. coli and transformed colonies are again obtained by plating on antibiotic plates. Individual colonies are picked and analyzed for the presence of the modification methylase. 8. If clones are found to express modification methylase, they are further analyzed for the simultaneous expression of the restriction endonuclease.
  • Methylase screening may be performed by:
  • the recombinant plasmid DNA molecule of the clone may be purified and exposed in vitro to the selecting restriction endonuclease to establish that it is resistant to digestion. Provided that the plasmid vector carries several sites for that endonuclease, resistance indicates modification rather than mutational site loss.
  • the total chromosomal DNA of the clone may be purified and exposed to the selective restriction endonuclease. If the clone carries the methylase gene, the bacterial chromosome should be fully methylated and, like the plasmid, should be found to be resistant to digestion.
  • the cell extract from the clone may be prepared and assayed in vitro for methylase activity (methylase protection and radioactive labelling).
  • Restriction endonuclease screening may be carried out as follows:
  • the cell extract from the clone may be prepared and assayed in vitro for its ability to digest substrate DNA, such as Ad-2. Cleavage of Ad-2 DNA indicates the presence of cloned restriction endonuclease.
  • the cells themselves may be tested in vivo for their ability to resist phage infection. Resistance to phage infection indicates the presence of the restriction endonuclease.
  • the restriction endonuclease used to selectively digest the plasmid library in step 6 is usually the same as that encoded by the restriction-modification system to be cloned.
  • the selective restriction endonuclease can be used to clone identi cal restriction-modification systems from other microorganisms, i.e., isoschizomers of the selective restriction endonuclease that cleave the same recognition sequence. It has been shown, for example, that Haelll was used to select the isoschizomeric restriction-modification system of BsuRI (Kiss et al., Nucleic Acid Res. 13:6403-6421 (1983)).
  • both the restriction endonuclease and modification methylase genes need not be maintained on the same cloning or expression vector within the same recombinant host.
  • the endonuclease gene may be located on one vector, while its corresponding methylase gene may be located on a separate compatible vector or located on the host genome.
  • Various combinations of maintaining both the modification and restriction genes within the same recombinant host can be constructed.
  • the only requirement, when cloning restriction endonuclease genes is that the recombinant host contain and express the methylase gene corresponding to the endonuclease gene being cloned.
  • Enhanced production of these enzymes can be accomplished, for example, by operably linking the desired gene(s) to a strong prokaryotic promoter.
  • Such promoters may be either constitutive or, more preferably, regulatable (i.e., indueible or derepressible).
  • constitutive promoters include the int promoter of bacteriophage ⁇ , and the bla promoter of the ⁇ -lactamase gene of pBR322, etc.
  • inducible prokaryotic promoters include the major left and right promoters of bacteriophage ⁇ (P L and P R ), the trp, recA, lacZ, gal, and tac promoters of E. coli, the ⁇ -amylase (Ulmanen, I., et al., J. Bacteriol. 162:176-182 (1985)), the ⁇ -28-specific promoters of B.
  • subtilis (Gilman, M.Z., et al., Gene 32:11-20 (1984)), the promoters of the bacteriophages of Bacillus (Gryczan, T.J., In: The Molecular Biology of the Bacilli, Academic Press, Inc., NY (1982)), and Streptomyces promoters (Ward, J.M., et al., Mol. Gen. Genet. 203:468-478 (1986)).
  • Prokaryotic promoters are reviewed by Glick, B.R., (J. Ind. Microbiol. 1:277-282 (1987)); Cenatiempo, Y. (Biochimie 68:505-516 (1986)); and Gottesman, S. (Ann. Rev. Genet.
  • restriction endonuclease gene may be cloned in a host which is not protected with the methylase gene, provided that the endonuclease gene is operably linked to a controllable promoter.
  • ribosome binding sites are disclosed, for example, by
  • the enzymes of this invention are preferably produced by fermentation of the recombinant host containing the cloned restriction endonuclease and modification methylase genes.
  • the recombinant host such as E. coli producing the cloned proteins, can be grown and harvested according to techniques well known in the art. Any nutrients that can be assimilated by the host containing the cloned restriction endonuclease and modification methylase genes may be added to the culture medium.
  • Glucose sucrose, maltose, lactose, glycerol, ethanol, lactates, various fats and oils, and others may be used as carbon source, while yeast extract, peptone, defatted soybeans, corn steep liquor, bouillon and others are suitable as nitrogen source.
  • Minerals and metal salts e.g., phosphates, potassium salts and magnesium salts, as well as vitamins and growth-promoting substances, may also be added as required.
  • Optimal culture conditions should be selected case by case according to the strain used and the composition of the culture medium. Restriction endonucleases and modification methylasesproduced by the recombinant hosts of this invention are accumulated inside the microbial cells.
  • Both the restriction endonuclease and modification methylase can be extracted and purified from the recombinant host by using known protein purification techniques commonly employed for these types of enzymes.
  • the crude lysate is adsorbed directly onto a Affigel blue agarose (Bio-rad Laboratories) column and eluted with 0 to 1.5 M potassium chloride solutions.
  • the active fractions collected are then absorbed again on blue agarose, followed by elution with 0 to 1.5 M potassium chloride solutions.
  • the active fractions were further purified through a Mono- Q column using a 0 to 1.0 M potassium chloride gradient to give a standard sample of NgoAIV.
  • the recombinant host containing the genes encoding for NgoAIV and M ⁇ NqoAIV was put on deposit on 21 February 1990 with the Patent Culture Collection, Northern Regional Research Center, USDA, 1815 N. University St., Peoria, IL 61604 USA, as deposit number NRRL B-18625.
  • Neisseria gonorrhoeae FA1090 Bacterial Strains and Growth Conditions Neisseria gonorrhoeae FA1090 (provided by Dr.
  • E. coli strains were grown at 37°C in YET broth (10 g/l Bacto trypton, 5 g/1 yeast extract and 5 g/l NaCL) with antibiotic supplements of ampicillin (Ap), 100 ⁇ g/ml; or tetracycline (Tc), 20 ⁇ g/ml as reguired.
  • E. coli strains, K802 Maniatis et al.: Molecular cloning. A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1982
  • DH5 ⁇ MCR were used interchangeably for cloning the restriction modification genes from Neisseria gonorrhoeae.
  • DH5 ⁇ MCR competent cells were obtain commercially from Life Technologies Inc., Gaithersburg, Maryland (LTI). Competent cells of K802 were made by a protocol previously described (Hanahan, D., J. Mol. Biol. 51:557-580 (1983)). EXAMPLE 2
  • Neisseria gonorrhoeae total genomic DNA was isolated by resuspending 2 grams of frozen cells in 8 mis of TNE buffer (50 mM Tris-HCl pH 8.0, 50 mM NaCl and 10 mM EDTA).
  • TNE buffer 50 mM Tris-HCl pH 8.0, 50 mM NaCl and 10 mM EDTA.
  • a 10 mg/ml lysozyme solution in TNE buffer was added to the cell suspension to a final concentration of 1 mg/ml. After a one hour incubation at 37°C, 10% SDS was added to a 2% final concentration and the suspension was shaken gently until lysis was complete. After cell lysis, the lysate was extracted once with phenol and twice with phenol:chloroform: isoamylalcohol (1:24:1).
  • DNA was spooled with a glass rod under two volumes of cold ethanol (-20°C), dissolved in TE (10 mM Tris-HCl pH 8.0, and 1 mM EDTA) and purified by CsCl-EtdBr gradient centrifugation.
  • Genomic DNA of Neisseria gonorrhoeae was digested partially with Hpall as follows: Purified genomic DNA was digested with Hpall in a 10 ⁇ l volume with 0.42, 0.21, 0.105, 0.0525, 0.026 or 0.013 u/ ⁇ g. Each digestion reaction contained 0.3 ⁇ g of DNA in 20 mM Tris-HCl (pH 7.4), and 10 mM MgC1 2 . After the samples were incubated one hour at 37°C, the DNA was analyzed by agarose gel electrophoresis.
  • Clal cleaved and dephosphorylated pCP13 vector was ligated with 2 ⁇ g of the partially digested genomic DNA using two units of T4 DNA ligase in 1 x ligase buffer (0.05 M Tris-HCl (pH 7.6), 10 mM MgC1 2 , 1 mM ATP, 1 mM DTT, and 5% (w/v) polyethylene glycol- 8000).
  • 1 ligase buffer 0.05 M Tris-HCl (pH 7.6), 10 mM MgC1 2 , 1 mM ATP, 1 mM DTT, and 5% (w/v) polyethylene glycol- 8000.
  • the 20 ⁇ l ligation reaction was incubated at room temperature (25°C) overnight.
  • ligated DNA 5 ⁇ l of the ligation reaction mixture
  • E. coli cells were infected with the packaging mix as follows: DH5 ⁇ MCR cells were prepared by growing an overnight culture in YET media containing 0.2% maltose. The next day, 500 ⁇ ls of these cells were inoculated into 10 mis of YET containing 0.2% maltose and grown to mid-log phase. These cells were then centrifuged and resuspended in 4.0 mis of sterile 10 mM MgSO 4 buffer.
  • 200 ⁇ ls of the cell suspension was mixed with 100 ⁇ ls of packaging mix. After a 30 minute incubation at 37°C without shaking, a 700 ⁇ l volume of SOC media (2% Bacto tryptone, 0.5% Yeast extract, 10 mM NaC1, 2.5 mM KC1, 10 mM MgC1 2 , 10 mM MgSO 4 and 20 mM glucose) was added. The cells were allowed to grow at 37°C in an air shaker-incubator for 30 minutes. The cells were then plated onto 9 YET agar plates containing tetracycline and incubated overnight.
  • SOC media 2% Bacto tryptone, 0.5% Yeast extract, 10 mM NaC1, 2.5 mM KC1, 10 mM MgC1 2 , 10 mM MgSO 4 and 20 mM glucose
  • Approximately 5 x 10 4 tetracycline resistant colonies were pooled together by scraping the cells from the agar surface. This was accomplished by flooding each plate with 2.5 mis of filter sterilized PEB I (50 mM glucose, 2.5 mM Tris-HCl pH 8.0 and 10 mM EDTA). After carefully resuspending the cells in buffer with a sterilized glass rod, the cell suspension was transferred to a sterile tube and stored at -70°C. Before freezing these cells, a 5 ml aliquot was removed and immediately inoculated into 1 liter of YET media containing tetracycline.
  • PEB I 50 mM glucose, 2.5 mM Tris-HCl pH 8.0 and 10 mM EDTA
  • Plasmid DNA was isolated from this cell suspension according to Example 2.
  • the isolated cosmid library was designated Neisseria gonorrhoeae plasmid library.
  • Neisseria gonorrhoeae plasmid library (5 ⁇ g) was digested in a reaction volume of a 100 ⁇ l containing 1 X REact 5 buffer (10 mM Tris-HCl pH 8.2, and 8 mM MgC1 2 ) with 70 units of Nael at 37°C for 4.5 hours.
  • One half of the digested DNA was dephosphorylated by adding 2 units of calf intestinal alkaline phosphatase (supplied by Boehringer Mannheim) to 50 ⁇ ls of the reaction mix. After a 1 hour incubation at 37°C, the DNA was extracted with an equal volume of phenyl:chloroform (1:1), ethanol precipitated, and resuspended in 10 ⁇ l of TE buffer.
  • E. coli DH5 ⁇ MCR competent cells were transformed with the digested DNA library according to the manufacturers suggested protocol. Briefly, 100 ⁇ ls of cold competent cells were mixed with the 10 ⁇ l sample of the Nael digested DNA. The cells were incubated without shaking for 30 minutes on ice. After a 45 second heat shock at 42°C, the cells were diluted with 900 ⁇ l of SOC and grown for 30 minutes at 37°C. Approximately 300 tetracycline resistant colonies were isolated after plating the transformed cells on YET agar plates containing tetracycline.
  • a 20 ml overnight culture was harvested and resuspended in 1 ml buffer containing 10 mM Tris-HCl (pH 7.5), 10 mM beta-mercaptoethanol and 1 mM EDTA.
  • Cells were sonicated on ice by 3 to 4, 10 second blast with a microtip probe. After sonication, the cell extract was centrifuged at 4°C for 30 sec. using a microfuge (1.5 ml tubes) in order to separate the cell debris.
  • Ad-2 DNA substrate (0.75 ⁇ g) was digested in 1 x REact 5 buffer with serial dilutions of extract as follows: Ad-2 DNA was diluted to a concentration of 0.038 ⁇ g/ul in 1 x REact 5 buffer. A 30 ⁇ l aliguot of the sample DNA was then added to the first tube and 20 ⁇ l aliguots were dispensed into the second, third and fourth tubes. A 3 ⁇ l volume of crude extract was mixed into the first tube. A 10 ⁇ l sample was then removed and serially diluted into the remaining tubes, with the final tube having the highest dilution of extract. The samples were incubated at 37°C for 1 hour and a 20 ⁇ l aliquot was analyzed by agarose gel electrophoresis.
  • Plasmid DNA was isolated from clone 12 using the small scale isolation procedure. Approximately 1 ⁇ g of this DNA was digested for 1 hour with 100 units of EcoRI in 100 ⁇ l of 1 X REact 3 buffer (50 mM Tris-HCl pH 8.0, 10 mM MgC1 2 and 0.1 M NaCl) at 37°C After incubation, the reaction mixture was extracted with phenol-chloroform, ethanol precipitated and dissolved in TE buffer.
  • 1 X REact 3 buffer 50 mM Tris-HCl pH 8.0, 10 mM MgC1 2 and 0.1 M NaCl
  • Competent DH5 ⁇ MCR cells were transformed with 3 ⁇ l of the ligation mixture according to the protocol described in Example 4. After the 30 minutes expression step, the cells were plated on YET plates containing ampicillin and incubated overnight.
  • NgoAIV restriction enzyme was purified from the overproducing strain, DH5ceMCR/pRMNgoAIV (example 6). Approximately 2,000 units of purified enzyme can be obtained from 4 grams of cells by following the procedure described below.
  • a 10 ml overnight culture of DH5 ⁇ MCR/pRMNgoAIV was grown in YET media containing ampicillin. The next day, one liter of fresh sterile media containing ampicillin was inoculated with 10 mis of the overnight culture and incubated at 37°C for 5 hours. A total of 8 grams wet cell weight were harvested from this culture.
  • One half of the cell pellet was resuspended in 8 mis of ice cold lysis buffer (20 mM Tris-HCl pH 7.6, 0.1 mM EDTA, 0.02% Na Azide, 0.02 mM PMSF, and 10mM Beta-mercaptoethanol) and sonicated on ice with 6 x 30 second blasts using a one half inch probe. Care was taken to keep the cell suspension ice cold during the sonication procedure.
  • the cell extract was then centrifuged at 4°C for 10 minutes using a microfuge (1.5 ml). Supernatant from the crude lysate was loaded directly onto a 3.5 ml blue agarose column (10 x 45 mm) equilibrated in buffer A (20 mM Tris-HCl pH 7.6, 0.1 mM EDTA, 0.02% Na Azide, and 0.2 mM PMSF). The column was washed with 10 x column volumes of buffer A. Then a 70 ml linear gradient of 0 to 1.5 M KCL in buffer B (20 mM Tris-HCl pH 7.6, 0.1 mM EDTA, and 0.2 mM PMSF) was applied and 0.5 ml fractions were collected. Fractions were tested for enzyme activity by incubating 1 ⁇ g Ad-2 DNA with a 1 ⁇ l aliquot of the fraction at 37°C for 1 hour. Fractions (42-57) exhibiting enzyme activity were pooled.
  • the three peak fractions exhibiting NgoAIV activity were pooled and dialyzed in buffer C containing 50% glycerol. Purified enzyme was stored at - 20°C.
  • the NgoAIV restriction enzyme purified as in example 7 was characterized to determine its nucleotide recognition sequence as well as the location of cleavage within this recognition site. As detailed below, NgoAIV was found to be a type II restriction enzyme which recognizes the palindromic sequence, 5' G ⁇ CCGGC3 ', and cleaves between the first G and C residues producing a 4-base 5' extension. The fragments produced from Ad-2 DNA by NgoAIV were identical to those produced by Nael, which recognizes the seguence 5' GCC ⁇ GGC 3'. In addition, fragment patterns of pBR322 digested with NgoAIV and Nael were identical. Attempts to clone Ad-2 DNA fragments generated by NgoAIV in an Nael digested vector were unsuccessful. These results suggested that unlike Nael, NgoAIV produces stick-ended fragments. Therefore, the cleavage site was determined.
  • the position of phosphodiester bond cleavage within the recognition site was determined by the method of Brown and Smith (Brown, N.L. et al., Methods. Enyzmol. 65:391-404 (1980)). Sequencing reactions were performed as described by Sanger et al. (J. Mol. Biol. 143:161-178 (1980)). Single-stranded M13mp19 DNA template containing an Nael site 60 bases from a primer (22 bases) were used to synthesize double-stranded DNA through the Nael site. The extended DNA was digested with Nael and NgoAIV separately. Aliguots of the digested products were run on a sequencing gel next to a sequencing ladder produced with the same primer and template.

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Abstract

Procédé pour l'expression d'une endonucléase de restriction qui reconnaît et sépare la séquence palindrome (I), entre les premiers résidus G et C à partir de l'extrémité 5', produisant une extension quatre base 5'. La présente invention porte également sur une méthylase de modification correspondant à l'endonucléase de restriction susmentionnée. La Neisseria gonorrhoeae est une source de ces enzymes. La présente invention a également trait au clonage et à l'expression des gènes codant pour cette endonucléase de restriction et cette méthylase de modification.
PCT/US1991/001599 1990-03-08 1991-03-08 CLONAGE ET EXPRESSION D'ENDONUCLEASES DE RESTRICTION DE $i(NEISSERIA) WO1991013975A1 (fr)

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Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
A.A. YANULAITIS, "Specificity of New Restrictases and Methylases. Unusual Modification of Cytosins in Position 4", Published 1984, by PLENUM PUBLISHING CORP., page 93-106, Translated from Mol. Discl. (Moscow), Vol. 18, Number 1, January/February 1983, pages 115-129. *
ABSTRACTS OF THE ASM ANNUAL MEETING, issued 1988, R.H. CHIEN et al., "Cloning and Characterization of a Neisseria Gonorrhoeae Strain MS11", see page 213, the Abstract K-41. *
NUCLEIC ACIDS RES., Volume 17, Number 8, issued 25 April 1989, A.L. HAMMOND et al., "Characterization of a Restriction Enzyme from a Strain of Neisseria Gonorrhoea which Recognizes 5'g ccggc3', an Isochizomer of Nael", see page 3320. *
NUCLEIC ACIDS RES., Volume 17, Suppl., issued 1989, R.J. ROBERTS, "Restriction Enzymes and their Isoschizomers", see page r362. *

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