# Patent

## Title
Method for cloning and expression of Acc65I restriction endonuclease and Acc65I methylase in E. Coli

## Abstract

An isolated DNA is provided which encodes a protein that is capable of binding to 5′GGTACC-3′, the isolated DNA being capable of hybridizing to SEQ ID NO:3 under stringent hybridization conditions. The isolated DNA may be alternatively characterized as coding for a protein having an amino acid sequence comprising SEQ ID NO:5 or by an amino acid sequence with an expectation value of less than E=e−02 in a BLAST search using SEQ ID NO:5. Vectors containing the isolated DNA and host cells expressing the vectors as well as a method for making recombinant Acc65I having the above properties are also provided.

## Background
RELATED APPLICATIONS
This application claims priority to U.S. provisional application Ser. No. 60/815,553 filed Jun. 14, 2006.


BACKGROUND OF THE INVENTION
Present embodiments of the invention relate to a Type II restriction endonuclease, Acinetobacter calcoaceticus 65 (Acc65I), obtainable from the recombinant strain carrying the genes for the Acc65I restriction-modification system from Acc65I in E. coli, and to a process for producing the same.
Restriction endonucleases are enzymes that occur naturally in certain unicellular microbes—mainly bacteria and archaea—and that function to protect these organisms from infections by viruses and other detrimental DNA elements. Restriction endonucleases bind to specific nucleotide (nt) sequences in double-stranded DNA molecules (dsDNA) and cleave the DNA molecules, often within or close to these sequences, fragmenting the molecules and triggering their ultimate destruction. Restriction endonucleases commonly occur with one or more companion enzymes termed modification methyltransferases. The methyltransferases bind to the same nt sequence in dsDNA as the restriction endonuclease, but instead of cleaving the DNA, they modify it by the addition of a methyl group to one of the bases in each strand of the sequence. This modification prevents the restriction endonuclease from binding to that site thereafter, effectively rendering the site resistant to cleavage. Methyltransferases act as cellular antidotes to the restriction endonucleases they accompany, protecting the DNA of the cell from destruction by its own restriction endonucleases. Together, a restriction endonuclease and its companion modification methyltransferase(s) form a restriction-modification (R-M) system, an enzymatic partnership that accomplishes for microbes, to some extent, what the immune system accomplishes for multicellular organisms.
A large and varied group of restriction endonucleases termed ‘type II’ cleave DNA at defined positions, and can be used in the laboratory to cut DNA molecules into precise fragments for gene cloning and analysis. The biochemical precision of type II restriction endonucleases far exceeds anything achievable by chemical methods, and so these enzymes have become the reagents sine qua non of modern molecular biology. They are the ‘scissors’ by means of which genetic engineering and analysis is performed, and their adoption has profoundly impacted the biomedical sciences over the past 25 years, transforming the academic and commercial arenas, alike. Their utility has spurred a continuous search for new restriction endonucleases, and a large number have been found. Today more than 200 Type II endonucleases are known, each possessing different DNA cleavage characteristics (Roberts and Macelis, Nucl. Acids Res. 29:268-269 (2001)); (REBASE®, http://rebase.neb.com/rebase). Concomitantly, the production and purification of these enzymes have been improved by the cloning and over-expression of the genes that encode them in non-natural production strain host cells such as E. coli. 
Since the various restriction enzymes perform similar roles in nature, and do so in much the same ways, it might be thought that they would resemble one another closely in amino acid sequence, organization, and behavior. Experience shows this not to be true, however. Surprisingly, far from resembling one another, most Type II restriction enzymes appear unique, resembling neither other restriction enzymes nor any other known kind of protein. Type II restriction endonucleases seem to have arisen independently of one another for the most part during evolution, and to have done so hundreds of times, so that today's enzymes represent a motley collection rather than a discrete family. Some restriction endonucleases act as homodimers, some as monomers, others heterodimers. Some bind symmetric sequences, others asymmetric sequences; some bind continuous sequences, others discontinuous sequences; some bind unique sequences, others multiple sequences. Some are accompanied by a single methyltransferase, others by two, and yet others by none at all. Often the methyltransferase is separate from, and functions independently of, the restriction endonuclease, but sometimes the two are fused and interdependent. When two methyltransferases are present, on some occasions they are separate proteins, on others they are fused. The orders and orientations of restriction and modification genes vary, with all possible organizations occurring. Several kinds of methyltransferases exist, some methylating adenines (m6A-MMases), others methylating cytosines at the N-4 position (m4C-MMases), or at the 5 position (m5C-MMases). Usually there is no way of predicting, a priori, which modifications will block a particular restriction endonuclease, which kind(s) of methyltransferases(s) will accompany that restriction endonuclease in any specific instance, nor what their gene orders or orientations will be.
From the point of view of cloning a Type II restriction endonuclease, the great variability that exists among restriction-modification systems means that, for experimental purposes, each is unique. Each enzyme is unique in amino acid sequence and catalytic behavior; each occurs in unique enzymatic association, adapted to unique microbial circumstances; and each presents the experimenter with a unique challenge. Sometimes a restriction endonuclease can be cloned and over-expressed in a straightforward manner but more often than not it cannot, and what works well for one enzyme can work not at all for the next. Success with one is no guarantee of success with another.
There is a continuing need for novel type II restriction endonucleases. Although type II restriction endonucleases that recognize a number of specific nucleotide sequences are currently available, new restriction endonucleases that recognize novel sequences provide greater opportunities and ability for genetic manipulation. Each new unique endonuclease enables scientists to precisely cleave DNA at new positions within the DNA molecule, with all the opportunities this offers.
SUMMARY OF THE INVENTION
In an embodiment of the invention, an isolated DNA coding for an endonuclease capable of binding 5′G/GTACC-3′ in a DNA substrate is provided which is characterized by SEQ ID NO:3, or by a DNA capable of hybridizing to SEQ ID NO: 3 under stringent hybridization conditions.
An example of the endonuclease is Acc65I restriction endonuclease that cleaves dsDNA between G and G as indicated below, producing DNA fragments with 4-nt cohesive ends.










5′ G/GTACC-3′




 



3′-CCATG/G-5′






An example of stringent hybridization conditions include 0.75M NaCl, 0.15 Tris, 10 mM EDTA, 0.1% Sodium Pyrophosphate, 0.1% SLS, 0.03% BSA, 0.03% Ficoll 400, 0.03% PVP and 100 μg/ml boiled calf thymus DNA at 50° C. for about 12 hours and washing 3 times for 30 minutes with 0.1×SET, 0.1% SDS, 0.1% sodium pyrophosphate and 0.1M phosphate buffer at 37° C.-55° C.
In another embodiment of the invention, an isolated protein is provided that encodes a protein comprising SEQ ID NO:5 or having an amino acid sequence identified by an expectation value of less than E=e−02 in a BLAST search using SEQ ID NO:5.
In another embodiment of the invention, a cloning vector is provided that includes the DNA described above and a host cell transformed by such cloning vector.
In another embodiment of the invention, a method is provided for producing recombinant Acc65I restriction endonuclease by culturing a host cell transformed with the vector described above under conditions suitable for expression of the endonuclease.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1. Cloned DBA sequence (SEQ ID NO:1) expressing Acc65I, the DNA derived from Acinetobacter calcoaceticus 65.
FIG. 2. Nucleotide sequences of a) the Acc65IM gene (SEQ ID NO:2), and b) the Acc651R gene (SEQ ID NO:3).
FIG. 3. Gene organization of a) the Acc65I restriction-modification system, b) the plasmid clone pCAB16-Acc65IM #5 carrying the Acc65I methyltransferase gene inserted into the BsaAI site of pCAB16, c) the plasmid clone pSX20-Acc65IM #4 (pSX20, a pBR322 derivative containing kanamycin marker) carrying the Acc65I methyltransferase gene inserted into the BamHI site of pSX20, and d) the plasmid clone pRRS-Acc65IR #6 carrying the Acc65I endonuclease gene inserted into the XbaI/XmaI site of pRRS (Skoglund et al. Gene 88(1):1-5 (1990).
FIG. 4. Predicted amino acid sequences of a) the M-Acc65I methyltransferase, and b) the R-Acc65I endonuclease.
FIG. 5. Restriction assay of NEB Strain #1686 E. coli ER2502 [pSX20-acc65IM #4, pRRS-acc65IR #6] (New England Biolabs, Inc. (NEB), Ipswich, Mass.).



DETAILED DESCRIPTION OF THE INVENTION
In embodiments of the invention, Acc65I is obtained by culturing transformed E. coli host cells, and recovering the endonuclease from the cells.
For recovering the enzyme of the present invention, E. coli may be grown using any suitable culturing technique. For example, E. coli may be grown in Luria broth media (BBL Microbiology Systems, Cockeysville, Md.) supplemented with 100 μg/ml ampicillin, 50 μg/ml kanamycin incubated aerobically at 37° C. Cells in the late logarithmic stage of growth are collected by centrifugation and either disrupted immediately or stored frozen at −70° C.
The Acc65I endonuclease can be isolated from bacterial host by conventional protein purification techniques. For example, cell paste is suspended in a buffer solution and ruptured by sonication, high-pressure dispersion or enzymatic digestion to allow extraction of the endonuclease by the buffer solution. Intact cells and cellular debris are then removed by centrifugation to produce a cell-free extract containing Acc65I. The Acc65I endonuclease is then purified from the cell-free extract by ion-exchange chromatography, affinity chromatography, molecular sieve chromatography, or a combination of these methods to produce the endonuclease of the present invention.
The details of cloning and expression of Acc65I is provided in the Examples. Alternatively, any person of ordinary skill in the art may generate the DNA sequences in FIG. 2 encoding the restriction endonuclease or methylase by commercial DNA synthesis technology or by PCR amplification using primers derived from the sequences in FIG. 2 and may additionally clone the synthesized DNA into a vector for expression in a competent host or by in vitro transcription/translation systems (U.S. Pat. Nos. 6,689,573; 6,905,837, 6,383,770).
The sequence for Acc65I methylase and restriction endonuclease are provided in FIG. 2. While Acc65IR has low homology to other restriction genes, variants of Acc65I are anticipated that retain Acc restriction activity and are intended to be included within the scope of the invention. For example, any DNA sequence encoding a protein having an amino acid sequence which matches the amino acid sequence of Acc65I in a blast search using an expectation value of less than E=e−02 is embodied in the invention (see international application PCT/US06/30419, incorporated herein by reference).
The cleavage characteristics of the Acc65I restriction endonuclease and its variants differ in an important way from those of isoschizomers such as KpnI. Acc65I cleaves DNA to produce fragments with 5′-extensions. KpnI, in contrast, cleaves to produce fragments with 3′-extensions. 5′-extensions are advantageous in that they can be filled-in by DNA polymerase to produce flush-ended fragments whose termini retain five of the six bases that comprise the Acc65I recognition sequence, that is to say 5′-GGTACC. If these filled-in fragments are ligated to other flush-ended fragments that bear a cytosine at their 5′-terminus, the Acc65I site, 5′-GGTACC, is restored at the junction. The ligated fragments can thus be re-cleaved with Acc65I; the site is not lost in the joining process. Furthermore, ligating filled-in Acc65I sites to one another produces a new sequence at the junction: 5′-GGTACGTACC (SEQ ID NO:6). This new sequence can no longer be cleaved by Acc65I (i.e., the Acc65I site is lost) but it can be cleaved by another restriction endonuclease, SnaBI, which recognizes the central 5′-TACGTA of the composite sequence. Finally, the termini of the DNA fragments produced by Acc65I are identical in sequence and polarity to those produced by several other restriction enzymes including BsiWI (recognition sequence: 5′-C/GTACG), BsrGI (recognition sequence 5′-T/GTACA), and TatI (recognition sequence 5′-W/GTACW). Consequently, DNA fragments produced by Acc65I will naturally anneal to and ligate with those produced by BsiWI, BsrGI and TatI.
Present embodiments of the invention are further illustrated by the following Examples. These Examples are provided to aid in the understanding of the invention and are not construed as a limitation thereof.
The references cited above and below are herein incorporated by reference.
EXAMPLE I
Cloning of the Acc65I Restriction-Modification Genes
1. Preparation of Genomic DNA
Genomic DNA was prepared from 10 g of Acinetobacter calcoaceticus 65, by the following steps:

    
    
        a. Cell wall digestion by addition of lysozyme (2 mg/ml final), sucrose (1% final), and 50 mM Tris-HCl, pH 8.0.
        b. Cell lysis by addition of 24 ml of Lysis mixture: 50 mM Tris-HCl pH 8.0, 62.5 mM EDTA, 1% Triton.
        c. Removal of proteins by phenol-CHCl3 extraction of DNA 2 times (equal volume).
        d. Dialysis in 4 liters of TE buffer, buffer change four times.
        e. RNase A treatment to remove RNA.
        f. Genomic DNA precipitation in 0.4M NaCl and 0.55 volume of 100% isopropanol, spooled, dried and resuspended in TE buffer.
    
    


2. Genomic DNA Digestion and Library Construction
Restriction enzymes ApoI, BglII, EcoRI, HindIII and Sau3AI were used to individually digest  ̃10 microgram quantities of Acinetobacter calcoaceticus 65 genomic DNA to achieve complete and partial digestions. Following heat-inactivation of the restriction enzymes at 65° C. for 15 minutes, the ApoI-digests were ligated to EcoRI-cleaved, bacterial alkaline phosphatase (BAP)-dephosphorylated pUC19 vector, the BglII-, and Sau3AI-digests were ligated to BamHI-cleaved, BAP-dephosphorylated pUC19, and HindIII-digests were ligated to HindIII-cleaved, BAP-dephosphorylated pUC19. The ligations, performed overnight with T4 DNA ligase, were then used to transform the endA− E. coli host, made competent by the CaCl2 method. Several thousand Ampicillin-resistant (ApR) transformants were obtained from each ligation. The colonies from each ligation were pooled and amplified in 500 ml LB+Ap overnight, and plasmid DNA was prepared from them by CsCl gradient purification to make primary plasmid libraries.
3. Cloning the Acc65I Genes by Methylase-Selection
One microgram of each of the primary plasmid libraries was challenged by digestion with  ̃25 units of Acc65I at 37° C. for 1 hr. One half microgram of each of the Acc65I-digested primary plasmid libraries was challenged by digestion with −5 units of lambda exonuclease at 37° C. for 30 min. followed by a heat inactivation at 65° C. for 15 min. The digestions were transformed back into E. coli and plated for survivors. 7 ApR survivors arose from the BglII-library, and 37, 12, 10, and 11 from the HindIII-, Sau3AI-, EcoRI-, and ApoI-libraries, respectively. Plasmids from BglII, HindIII, Sau3AI, EcoRI, and ApoI survivors were extracted using mini-preps, and subjected to Acc65I-digestion. 3 of the 10 clones from the EcoRI-library were found to be resistant to Acc65I, but all those from BglII-, HindIII-, Sau3AI-, and ApoI-libraries were found to be sensitive. Plasmid DNA from survivors from the EcoRI-library was prepared by CsCl gradient purification. The individual clones from the EcoRI-library were inoculated into 50 ml LB+ApR and grown at 37° C. overnight. All EcoRI-library clones did not express detectable Acc65I endonuclease activity.
4. Identification of the Acc65IRM Restriction-Modification Genes
The nucleotide sequence of the inserted DNA in the Acc65I-resistant plasmid clones from the EcoRI-library was determined by dideoxy-automated sequencing. Transposon-insertion into clone EcoRI using the NEB's GPS-1 System (Ipswich, Mass.), provided the initial substrates for sequencing. Inverse PCR, and primer-walking was used subsequently, on clones EcoRI, and inverse-PCR generated clones of Acinetobacter calcoaceticus 65 genomic DNA, to finalize the sequence. A total of 5812 bp was determined (FIG. 1), within which two complete open reading frames (ORFs) of 1271 bp (complement nt 1578-2849), and 1619 bp (nt 2906-4525) were found (FIG. 2). The two ORFs have diverging orientation and are separated by 32 bp (FIG. 3). Analysis of the ORFs indicated that the smaller ORF, subsequently termed Acc65IM, encodes the modification enzyme, M-Acc65I, and the larger ORF, subsequently termed Acc65IR, the R-Acc65I restriction enzyme. M-Acc65I is predicated to be 422 aa in length and to have a molecular mass of 48,398 Daltons (or 422 aa and 48,267 Daltons, without the N-terminal fMet). R-Acc65I is predicted to be 539 aa in length and to have a molecular mass of 61,221 Daltons (539 aa and 61,221 Daltons without the fMet) (FIG. 4).
The RM-Acc65I restriction-modification system appears to comprise a separate DNA-restriction gene (Acc65IR) and a DNA-methylation gene (Acc65IM). M-Acc65I includes amino acid sequence motifs characteristic of the beta-class of DNA-adenine methyltransferases including . . . DPPY . . . (aa 80-83), . . . VLDFFAGSATT . . . (SEQ ID NO:7) (aa 357-367). M-Acc65I displays substantial homology to the KpnI modification enzyme, and to several similar putative M-subunits in Genbank.
R-Acc65I shows little homology to any other protein in Genbank. This is typical of restriction endonucleases of type II R-M systems.
EXAMPLE II
Cloning of Acc65I Methylase and Endonuclease in E. coli 
The Acc65IM gene was amplified by PCR from genomic DNA. Following purification, the resulting PCR fragment was blunt-end ligated into the BsaAI site of pCAB16, and transformed into E. coli. pCAB16 is a pUC18 derivative containing the mspIR gene in the polylinker of pUC18 in line with the Plac promoter. pCAB16 contains a single BsaAI site within the mspIR gene. Insertions at this site interrupt mspIR expression (which would otherwise be lethal) enabling plasmids containing inserts to be selectively recovered with high efficiency. Plasmid DNA of pCAB16-Acc65IM #5 containing the Acc65IM PCR insert was prepared by CsCl gradient purification (FIG. 3). The Acc65IM gene was purified from a BamHI-digested pCAB16-Acc65IM plasmid by gel purification. The resulting DNA fragment was ligated into BamHI-, BAP-dephosphorylated pSX20 and transformed into E. coli (FIG. 3). Clones carrying the PCR insert were completely resistant to Acc65I digestion. An E. coli host containing a pSX20-Acc65IM plasmid was made competent by CaCl2 method.
The Acc65IR gene was amplified by PCR from genomic DNA. Following purification, the resulting PCR fragment was digested with XbaI+XmaI and ligated into XbaI-, XmaI-digested PRRS and transformed into E. coli host containing the pSX20-Acc65IM plasmid (FIG. 3).
EXAMPLE III
Production of Recombinant Acc65I Endonuclease
166 grams of E. coli cell pellet were suspended in 1 liter of Buffer A (20 mM Tris-HCl (pH 7.4), 10 mM 2-mercaptoethanol, 5% Gycerol) containing 200 mM NaCl, and passed through a Gaulin homogenizer at  ̃12,000 psig. The lysate was centrifuged at  ̃13,000×G for 40 minutes and the supernatant collected.
The supernatant solution was applied to a 372 ml Heparin Hyper-D column (Biosepra, Marlborough, Mass.) which had been equilibrated in Buffer A containing 200 mM NaCl. A 940 ml wash of buffer A containing 200 mM NaCl was applied, then a 2.2 L gradient of NaCl from 0.2M to 1M in buffer A was applied and fractions were collected. Fractions were assayed for Acc65I endonuclease activity by incubating with 1 microgram of pBC4 plasmid DNA (dam-, dcm-) (NEB#N0354S) in 50 microliters containing NEBuffer 3, supplemented with 100 μg/ml BSA for 5 minutes at 37° C. (NEB, Ipswich, Mass.). Acc65I activity eluted at 0.5M to 0.6M NaCl.
The Heparin Hyper-D column fractions containing the Acc65I activity were pooled and loaded directly onto a 63 ml Ceramic HTP column (Biosepra, Marlborough, Mass.) equilibrated in Buffer A containing 200 mM NaCl. A 160 mL wash of buffer A, containing 200 mM NaCl, was applied, then a 630 mL gradient of KHPO4 (pH 7.0) from 0M to 0.55M in buffer A, containing 200 mM NaCl, was applied and fractions were collected. Fractions were assayed for Acc65I endonuclease activity by incubating with 1 microgram of pBC4 plasmid DNA (dam-, dcm-) (NEB#N0354S, NEB, Ipswich, Mass.) in 50 microliters NEBuffer 3, supplemented with 100 μg/ml BSA for 5 minutes at 37° C. Acc65I activity eluted at 0.2M to 0.3M KHPO4 (NEB, Ipswich, Mass.).
The Ceramic HTP column fractions containing the Acc65I activity were pooled and dialyzed into Buffer A, containing 200 mM NaCl (Biosepra, Marlborough, Mass.). This pool was loaded onto a 80 ml Q Sepharose column which had been equilibrated in buffer A containing 0.2M NaCl. A 130 mL wash of buffer A containing 200 mM NaCl was applied, then a 680 ml gradient of NaCl from 0.2M to 1M in buffer A was applied and fractions were collected. Fractions were assayed for Acc65I endonuclease activity by incubating with 1 microgram of pBC4 plasmid DNA (dam-, dcm-) (NEB#N0354S) in 50 microliters NEBuffer 3, supplemented with 100 μg/ml BSA for 5 minutes at 37° C. (NEB, Ipswich, Mass.). Acc65I activity eluted at 0.28M to 0.36M NaCl.
The Q Sephrarose column fractions containing the Acc65I activity were pooled and dialyzed into Buffer A, containing 200 mM NaCl. This pool was loaded onto a 21 ml Heparin TSK column which had been equilibrated in buffer A containing 0.2M NaCl. A 60 mL wash of buffer A containing 200 mM NaCl was applied, then a 200 ml gradient of NaCl from 0.2M to 1M in buffer A was applied and fractions were collected. Fractions were assayed for Acc65I endonuclease activity by incubating with 1 microgram of pBC4 plasmid DNA (dam-, dcm-) (NEB#N0354S) in 50 microliter NEBuffer 3 (NEB, Ipswich, Mass.), supplemented with 100 μg/ml BSA for 5 minutes at 37° C. Acc65I activity eluted at 0.4M to 0.48M NaCl.
The pool was dialyzed into Storage Buffer (10 mM KHPO4 pH 7.0, 100 mM NaCl, 11.0 mM DTT, 0.1 mM EDTA, 50% Gycerol). Eighty million units of Acc65I were obtained from this procedure. The Acc65I endonuclease thus produced was substantially pure and free of contaminating nucleases.
Activity Determination
Acc65I activity: Samples of 1 microliter were added to substrate solutions consisting of 1×NEBuffer 3 containing 1 microgram of pBC4 DNA (dam-, dcm-) (NEB#N0354S)/50 microliters, supplemented with 100 μg/ml BSA (NEB, Ipswich. MA). The reaction was incubated at 37° C. for 60 minutes. The reaction was terminated by adding 20 microliter of stop solution (50% glycerol, 50 mM EDTA pH 8.0, and 0.02% Bromophenol Blue (Sigma, St. Louis, Mo.) The reaction mixture was applied to a 1% agarose gel and electrophoresed. The bands obtained were identified by comparison with DNA size standards.
Unit Definition: One unit of Acc65I is defined as the amount of Acc65I required to completely cleave one microgram of pBC4 plasmid DNA in a reaction volume of 50 microliters of 1×NEBuffer 3 supplemented with 100 μg/ml BSA, within one hour at 37° C. (NEB, Ipswich, Mass.).
EXAMPLE IV
Expression of Acc65I Endonuclease in E. coli 
The plasmid [pRRS-Acc65IR XbaI/XmaI] was transferred into E. coli containing compatible plasmid [pSX20-Acc65IM BamHI #4] and plated on ApR+KnR plates at 37° C. overnight. Several individual colonies were inoculated into 50 ml LB+ApR+KnR and grown at 37° C. overnight. All clones expressed Acc65I endonuclease activity at >106 μ/g per gram of wet E. coli cells.

## Claims

1. An isolated DNA coding for a protein comprising SEQ ID NO: 5.

2. A cloning vector comprising the isolated DNA of claim 1.

3. A host cell transformed by the cloning vector of claim 2.

4. A method of producing recombinant Acc65I restriction endonuclease by culturing a host cell transformed with the vector in claim 2 under conditions suitable for expression of the restriction endonuclease.