# Patent

## Title
Method for cloning and expression of AcuI restriction endonuclease and AcuI methylase in E. coli

## Abstract

The present invention relates to recombinant DNA encoding the AcuI restriction endonuclease as well as AcuI methylase, and expression of AcuI restriction endonuclease and AcuI methylase in E. coli cells containing the recombinant DNA.

## Background
BACKGROUND OF THE INVENTION
The present invention relates to recombinant DNA that encodes the AcuI restriction endonuclease (AcuI endonuclease or AcuIR) as well as the AcuI methyltransferase (AcuI methylase or M.AcuI), and expression of AcuI endonuclease and methylase in E. coli cells containing the recombinant DNA. AcuI is an isoschizomer of Eco57I (MBI Fermentas (Vilnius, Lithuania) product #ER0341).
Type II restriction endonucleases are a class of enzymes that occur naturally in bacteria and in some viruses. When they are purified away from other bacterial/viral proteins, restriction endonucleases can be used in the laboratory to cleave DNA molecules into small fragments for molecular cloning and gene characterization.
Restriction endonucleases recognize and bind to particular sequences of nucleotides (the ‘recognition sequence’) along DNA molecules. Once bound, they cleave the molecule within (e.g. BamHI), to one side of (e.g. SapI), or to both sides (e.g. TspRI) of the recognition sequence. Different restriction endonucleases have affinity for different recognition sequences. At least two hundred and forty restriction endonucleases with unique specificities have been identified among the many hundreds of bacterial species that have been examined to date (Roberts et al., Nucl. Acids Res. 31:418-420 (2002)).
Restriction endonucleases typically are named according to the bacteria from which they are discovered. Thus, the species Deinococcus radiophilus for example, produces three different restriction endonucleases, named DraI, DraII and DraIII. These enzymes recognize and cleave the sequences 5′TTT/AAA3′, 5′RG/GNCCY3′ and 5′CACNNN/GTG3′ respectively. Escherichia coli RY13, on the other hand, produces only one enzyme, EcoRI, which recognizes the sequence 5′G/AATTC3′.
A second component of bacterial/viral restriction-modification (R-M) systems is the methylase. These enzymes co-exist with restriction endonucleases and they provide the means by which bacteria are able to protect their own DNA and distinguish it from foreign DNA. Modification methylases recognize and bind to the same recognition sequence as the corresponding restriction endonuclease, but instead of cleaving the DNA, they chemically modify one particular nucleotide within the sequence by the addition of a methyl group to produce C5 methyl cytosine, N4 methyl cytosine, or N6 methyl adenine. Following methylation, the recognition sequence is no longer cleaved by the cognate restriction endonuclease. The DNA of a bacterial cell is always fully modified by the activity of its modification methylase. It is therefore completely insensitive to the presence of the endogenous restriction endonuclease. Only unmodified, and therefore identifiable foreign DNA, is susceptible to restriction endonuclease recognition and cleavage. During and after DNA replication, usually hemi-methylated DNA (DNA methylated on one strand) is also resistant to the cognate restriction endonuclease.
With the advancement of recombinant DNA technology, it is now possible to clone genes and overproduce the enzymes in large quantities. The key to isolating clones of restriction endonuclease genes is to develop an efficient method to identify such clones within genomic DNA libraries, (i.e. populations of clones derived by ‘shotgun’ procedures) when they occur at frequencies as low as 10−3 to 10−4. Preferably, the method should be selective, such that the unwanted clones with non-methylase inserts are destroyed while the desirable rare clones survive.
A large number of type II restriction-modification systems have been cloned. The first cloning method used bacteriophage infection as a means of identifying or selecting restriction endonuclease clones (EcoRII: Kosykh et al., Mol. Gen. Genet. 178:717-719, (1980); HhaII: Mann et al., Gene 3:97-112, (1978); PstI: Walder et al., Proc. Nat. Acad. Sci. 78:1503-1507, (1981)). Since the expression of restriction-modification systems in bacteria enables them to resist infection by bacteriophages, cells that carry cloned restriction-modification genes can, in principle, be selectively isolated as survivors from genomic DNA libraries that have been exposed to phage. However, this method has been found to have only a limited success rate. Specifically, it has been found that cloned restriction-modification genes do not always confer sufficient phage resistance to achieve selective survival.
Another cloning approach involves transferring systems initially characterized as plasmid-borne into E. coli cloning vectors (EcoRV: Bougueleret et al., Nucl. Acids. Res. 12:3659-3676 (1984); PaeR7: Gingeras and Brooks, Proc. Natl. Acad. Sci. USA 80:402-406 (1983); Theriault and Roy, Gene 19:355-359 (1982); PvuII: Blumenthal et al., J. Bacteriol. 164:501-509 (1985); Tsp45I: Wayne et al. Gene 202:83-88 (1997)).
A third approach is to select for active expression of methylase genes (methylase selection) (U.S. Pat. No. 5,200,333 and BsuRI: Kiss et al., Nucl. Acids. Res. 13:6403-6421 (1985)). Since restriction-modification genes are often closely linked together, both genes can often be cloned simultaneously. This selection does not always yield a complete restriction system however, but instead yields only the methylase gene (BspRI: Szomolanyi et al., Gene 10:219-225 (1980); BcnI: Janulaitis et al., Gene 20:197-204 (1982); BsuRI: Kiss and Baldauf, Gene 21:111-119 (1983); and PstI: Walder et al., J. Biol. Chem. 258:1235-1241 (1983)).
A more recent method, the “endo-blue method”, has been described for direct cloning of thermostable restriction endonuclease genes into E. coli based on an indicator strain of E. coli containing the dinD::IacZ fusion (U.S. Pat. No. 5,498,535; Fomenkov et al., Nucl. Acids Res. 22:2399-2403 (1994)). This method utilizes the E. coli SOS response signal following DNA damage caused by restriction endonucleases or non-specific nucleases. A number of thermostable nuclease genes (TaqI, Tth111I, BsoBI, Tf nuclease) have been cloned by this method (U.S. Pat. No. 5,498,535). The disadvantage of this method is that some positive blue clones containing a restriction endonuclease gene are difficult to culture due to the lack of the cognate methylase gene.
There are three major groups of DNA methyltransferases based on the position and the base that is modified (C5-cytosine methylases, N4-cytosine methylases, and N6-adenine methylases). N4-cytosine and N6-adenine methylases are amino-methyltransferases (Malone et al. J. Mol. Biol. 253:618-632 (1995)). When a restriction site on DNA is modified (methylated) by the methylase, it is resistant to digestion by the cognate restriction endonuclease. Sometimes methylation by a non-cognate methylase can also confer DNA sites resistant to restriction digestion. For example, Dcm methylase modification of 5′ CCWGG 3′ (W=A or T) can also make the DNA resistant to PspGI restriction digestion. Another example is that CpG methylase can modify the CG dinucleotide of the NotI site (5′ GCGGCCGC 3′) and make it refractory to NotI digestion (New England Biolabs' (Beverly, Mass.) catalog, 2002-03, page 252). Therefore methylases can be used as a tool to modify certain DNA sequences and make them uncleavable by restriction enzymes.
Type II methylase genes have been found in many sequenced bacterial genomes (GenBank, http://www.ncbi.nlm.nih.gov; and Rebase®, http://rebase.neb.com/rebase). Direct cloning and over-expression of ORFs adjacent to methylase genes yielded restriction enzymes with novel specificities (Kong et al. Nucl. Acids Res. 28:3216-3223 (2000)). Thus microbial genome mining emerged as a new way of screening/cloning new type II restriction enzymes and methylases and their isoschizomers.
Because purified restriction endonucleases and modification methylases are useful tools for creating recombinant DNA molecules in the laboratory, there is a strong commercial interest to obtain bacterial strains through recombinant DNA techniques that produce large quantities of restriction enzymes and methylases. Such over-expression strains should also simplify the task of enzyme purification.
AcuI recognizes the double-stranded DNA sequence 5′CTGAAG3′ (or 5′CTTCAG3′ bottom strand) and cleaves 16/14 bases downstream of its recognition sequence to generate a 2-base 3′ cohesive end. AcuI is classified as a type IIs restriction enzyme since it cleaves DNA downstream from its recognition site. In addition, AcuI was expected to be a type IIG enzyme as it is an isoschizomer of Eco57I, the first such restriction enzyme to be identified (Janulaitis et al. Nucl. Acids. Res. 20:6043-6049 (1992)). Type IIG restriction endonucleases are distinguished by the fact that they possess both restriction and modification activity in one polypeptide chain (Pingoud and Jeltsch, Nucl. Acids Res. 29:3705-3727 (2001)). Therefore, when such an enzyme is employed in vitro to digest DNA, two competing activities are at work. If the modification (methylation) activity is significant, some of the substrate recognition sites may become modified before the endonuclease function is complete. This outcome is clearly apparent when using Eco57I to cleave lambda DNA, for example. (see FIG. 1). In contrast, when native purified AcuI is used to cleave the same substrate, complete digestion is observed. Therefore, an attempt was made to clone the AcuI restriction-modification system into E. coli in order to over-express and purify commercial quantities of the AcuI restriction endonuclease.
SUMMARY OF THE INVENTION
The present invention relates to a method for cloning the AcuI restriction endonuclease and AcuI methylase genes into E. Coli by methylase selection and inverse PCR amplification of the DNA adjacent to the AcuI methylase gene. AcuI endonuclease is native to the bacterium Acinetobacter calcoaceticua SRW4 (New England Biolabs strain #3307). Genomic DNA was isolated from this strain and several genomic DNA libraries were prepared. After digesting the libraries with native AcuI, the putative AcuI methylase gene was selected. The putative gene displayed high sequence similarity to the amino-methyltransferase family and notably to the eco57IM gene.
Assuming that the AcuI R-M gene organization would be similar to that of Eco571, inverse PCR was performed to locate the AcuI endonuclease gene downstream from the methylase gene. A BLAST search of the downstream region revealed that the downstream DNA was homologous to the Acinetobacter ADP1 mismatch repair gene mutS. Therefore, inverse PCR efforts were redirected to the region upstream of the putative acuIM gene. After walking 3.0 kb upstream, the acuIRM gene was identified. Herein, acuIRM refers to the gene encoding the AcuI endonuclease-methylase fusion protein. Two rounds of inverse PCR were necessary to completely identify the acuIRM gene as the open reading frame is 3003 bp, which encodes a protein of 1000 amino acids with a calculated molecular weight of 115,826 daltons. As well, the acuIM open reading frame is relatively large at 1608 bp, encoding a protein of 535 amino acids with a calculated molecular weight of 61,458 daltons.
Construction of an AcuI overexpression strain proved to be very difficult due to the large size of the AcuI endonuclease gene and gene product. First of all, PCR amplification of a 3.0 kb gene is subject to the limitations of polymerase fidelity and processivity. After initially failing to isolate an active clone, KOD HiFi polymerase (Novagen (Madison, Wis.)) was employed to increase the probability of cloning a wild-type acuIRM gene. Secondly, over-expression of such a large polypeptide is limited in prokaryotic hosts such as E. coli. Furthermore, type IIG restriction enzymes possess relatively low specific activity so detection of an active enzyme within a cellular extract may be a limiting factor in the isolation of a recombinant clone. AcuI over-expression was attempted from a total of three vectors. Finally, a wild-type recombinant AcuI clone was isolated using pET28a as the expression vector and ER2744 as the T7 expression host. In order to premodify host ER2744, a 2.8 kb SalI fragment from an original methylase clone (ANS6) was subcloned into pACYC184 to create pACYC184-AcuIM clone #9. Therefore, the final over-expression strain was ER2744 [pET28a-AcuIRM, pACYC184-AcuIM]. This strain (NEB#1513) produces 17,400 units per gram of wet cells.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 Digestion properties of native AcuI as compared to recombinant Eco57I (Fermentas (Vilnius, Lithuania) product #ER0341). Five units of each enzyme were used to digest 1 μg of lambda DNA for 0.5, 1, 2 or 16 hours at 37° C. in the presence of 0.08 mM S-adenosylmethionine. An arrow indicates the incomplete digestion product remaining after incubation with Eco57I.
FIG. 2 Gene organization of the AcuI R-M system relative to the gene organization of the Eco57I R-M system. acuIRM, AcuI restriction endonuclease gene; acuIM, AcuI methylase gene; eco57IRM, Eco57I restriction endonuclease gene; eco57IM, Eco57I methylase gene
FIG. 3 AcuI methylase gene sequence (SEQ ID NO:1) (acuIM, 1608 bp) and the encoded amino acid sequence (SEQ ID NO:2).
FIG. 4 AcuI endonuclease gene sequence (SEQ ID NO:3) (acuIRM, 3003 bp) and the encoded amino acid sequence (SEQ ID NO:4).
FIG. 5 Recombinant AcuI endonuclease activity in cell extract. (U) is undigested lambda substrate; (N) is digestion with 5 units native AcuI.



DETAILED DESCRIPTION OF THE INVENTION
The method described herein by which the AcuI methylase and the AcuI endonuclease genes are preferably cloned and expressed in E. coli employs the following steps:
1. Preparation of Genomic DNA and Construction of Plasmid Libraries to Select a Functional AcuI Methylase Gene
The first step in creating the recombinant AcuI R-M system in E. coli was to isolate a functional methylase gene in order to pre-protect the host genomic DNA against expression of the AcuI endonuclease gene. Vector pRRS (Skoglund et al. Gene 88: 1-5 (1990)) was linearized with either BamHI, EcoRI or SphI followed by CIP treatment. AcuI genomic DNA was purified from Acinetobacter calcoaceticua SRW4 (New England Biolabs strain #3307) by the standard procedure. AcuI genomic DNA (9 μg per reaction) was completely digested with BamHI, EcoRI or SphI or partially digested with ApoI, Sau3AI or NlaIII. The partial digests were optimized to yield DNA fragments in the 2-10 kb range. After gel purification of the six fragment types, each lot was ligated into vector pRRS. The BamHI and Sau3AI fragments were ligated into BamHI cut pRRS. The EcoRI and ApoI fragments were ligated into EcoRI cut pRRS. And the SphI and NlaIII fragments were ligated into SphI cut pRRS. The six ligation reactions were incubated overnight and then each was transformed into ER2502 by electroporation. Each transformation yielded >20,000 colonies. The colonies from the partial libraries were combined into one 500 mL culture and the colonies from the complete libraries were combined into a second 500 mL culture. After overnight incubation, plasmid DNA was prepared from each by the Qiagen (Studio City, Calif.) Maxiprep procedure. Fifty micrograms of each library (partial versus complete) were digested overnight with native AcuI. A sample of each digest was then analyzed by agarose gel electrophoresis and two shortcomings were revealed. First, the overnight digest was incomplete (due to excessive DNA) so this critical “challenge” step was unsuccessful. But most importantly, the partial AcuI digest revealed that a majority of the library DNA clones did not contain genomic DNA inserts. This outcome was most likely due to inadequate CIP treatment of vector PRRS. Therefore, new vector was prepared but pUC19 was substituted in place of pRRS. Vector pUC19 possesses two AcuI (Eco57I) sites, a necessary feature for the methylation selection procedure. Ten micrograms of pUC19 were digested with BamHI, EcoRI or SphI for 2 hrs at 37° C. Next, 5 units of CIP were added to each reaction for 30 min at 37° C. followed by heat-inactivation for 30 min at 65° C. Finally, the pUC19 vector DNA was purified by a Qiagen (Studio City, Calif.) miniprep spin column. The three vectors types were ligated with the six genomic DNA types as described above to create three partial and three complete AcuI genomic DNA libraries. The pUC19 ligations each yielded >30,000 colonies after transformation into ER2502 by electroporation (except the BamHI ligation yielded only 18,000 colonies). An aliquot of each ligation was separately transformed into ER2683 to conduct blue/white screening to assess the number of insert-containing clones. The percentage of inserts in each library varied from 50-87% (except the BamHI assessment found only 1 of 13 white colonies). Therefore, the BamHI library was excluded. The “complete” library was then created by inoculating approximately 10,000 colonies of each of the EcoRI and SphI libraries into 500 mL LB plus Amp, growing for 3 hours and isolating plasmid DNA by the Qiagen (Studio City, Calif.) Maxiprep procedure. The “partial” library was created in the same manner by pooling approximately 10,000 ApoI colonies, 10,000 NlaIII colonies and 20,000 Sau3AI colonies.
Each library (5 μg) was challenged with 20 units of native AcuI overnight at 37° C. in a 250 μL reaction. After heat inactivation of AcuT, small aliquots (1, 2, 5, and 10 μL) were transformed into ER2502 and plated on ampicillin agar plates. Each transformation yielded 5-20 colonies after incubation at 37° C. overnight. Nine colonies from the partial library and nine colonies from the complete library were grown for 6 hrs and plasmid DNA was prepared. The eighteen clones were digested with 4 units of native AcuI to test for the presence of a functional AcuI methylase gene. Three clones were protected from AcuI digestion and thus potentially carried the AcuI methylase gene. These clones designated ANS3, ANS6 and ANS8 were sequenced with pUC19 primers s1233s and s1224s (New England Biolabs, (Beverly, Mass.)) to reveal a partial open reading frame with similarity to the eco57IM gene. Clone ANS6 was chosen for further characterization and the 2.8 kb gene insert was confirmed to encode the AcuI methylase gene (acuIM). The vector/insert junction revealed that clone ANS6 was isolated from the NlaIII partial library. Methylase clone ANS6 was completely sequenced by primer walking to define a 1608 bp ORF that encodes a protein of 535 amino acids.
2. Cloning of the AcuI Restriction Endonuclease Gene (acuIRM) by Inverse PCR.
An assumption was made that the AcuI R-M gene organization would be the same as the Eco57I R-M system. Therefore, inverse PCR was initially conducted downstream of the AcuI methylase gene in order to locate the AcuI endonuclease gene. Inverse PCR primers were designed to anneal to the end of insert ANS6. AcuI genomic DNA was digested with the following enzymes: AciI, AflIII, AluI, BsaAI, MfeI, MseI, RsaI, Sau3AI and Tsp45I. After self-ligation of the digestion products, inverse PCR was performed using 35 cycles. Inverse PCR products were obtained from all nine of the digestion/ligation reactions. The AflIII inverse PCR product (1.6 kb) was gel-purified and was partially sequenced. A BLAST search of this sequence indicated a high degree of similarity to the Acinetobacter ADP1 mismatch repair gene mutS. Therefore, it was concluded that acuIRM does not reside immediately downstream of acuIM.
Inverse PCR efforts were then redirected to the region upstream of acuIM. The first round of upstream inverse PCR (using 8 different digests) yielded products ranging from 0.4-1.8 kb. The complete 1.8 kb region was sequenced to reveal an incomplete open reading frame with high similarity to the Eco57I endonuclease gene. This partial ORF was then assumed to be acuIRM. As this gene was expected to be approximately 3.0 kb, a second round of inverse PCR was performed (using 8 different digests). Round two PCR products ranged from 0.6-2.0 kb. After multiple sequencing reactions, the beginning of acuIRM was found as an in-frame GTG was located immediately following a TAA stop codon. The GTG codon codes for valine and translation can be initiated at this codon when other essential genetic elements are present. Initially, the acuIRM ORF was defined as 2961 bp due to an erroneous sequence at the 3′ end of the gene. Later, the corrected acuIRM ORF was confirmed to be 3003 bp.
3. Attempt to Over-Express acuIRM from pACYC-T7ter
The methylase clone ANS6 was transformed into T7 expression strain ER2744 to prepare pre-modified host cells. The initially defined acuIRM gene (2961 bp) was PCR-amplified from AcuI genomic DNA using a forward primer (291-043) with an NdeI site overlapping the GTG start to create an ATG start and a reverse primer (291-044) encoding a BamHI site downstream of the erroneous stop codon. A 3.0 kb PCR product was obtained using a Taq/Vent® polymerase mix (50:1 units, respectively). After NdeI/BamHI digestion and gel-purification, the fragment was ligated into NdeI/BamHI cut, CIP-treated pACYC-T7ter. This expression vector is derived from pACYC184 and is present at 5-8 copies in the cell (see U.S. Pat. No. 6,335,190). The ligation reaction was transformed into ER2744 [pUC19-AcuIM clone ANS6] by electroporation. Eighteen colonies were grown for plasmid DNA isolation and induction with IPTG. None of the induced cultures displayed AcuI endonuclease activity when cell extract was incubated with substrate pUC19. Failure to obtain an over-expressing clone may have been due to any of the following reasons: A) All eighteen clones may have contained detrimental mutations as a result of PCR amplification by Taq/Vent® polymerase. B) The low copy number of the expression vector results in an undetectable level of AcuI endonuclease. C) Unknown at the time, amplification with reverse primer 291-044 results in a modified gene product where the last five amino acids are altered.
4. Attempt to Over-Express acuIRM from pUC19-Kan
To increase the probability of isolating a recombinant AcuI clone, items A and B were addressed. First, the high-copy number vector pUC19-Kan was chosen for endonuclease expression. An additional benefit of employing a kanamycin vector is reduced loss of plasmid in late-log cultures as compared to ampicillin selection (see pET system manual, www.novagen.com) To address the potential problem of polymerase fidelity, PCR-amplification of the acuIRM ORF was attempted with Vent® and Deep Vent® (New England Biolabs (Beverly, Mass.)) with negative results. Consequently, the acuIRM ORF was amplified with a Taq/Vent® mix (as described previously) using forward primer 291-287 and reverse primer 291-044. Primer 291-287 contains a PstI site, a Shine-Delgarno sequence and an ATG start. After PstI digestion and gel-purification, the acuIRM PCR product was phosphorylated with T4 polynucleotide kinase. Next, the acuIRM gene was ligated into pUC19-Kan which had been prepared by PstI/HincII digestion, CIP treatment and gel-purification. The ligation reaction was transformed into ER2744 [pACYC184-AcuIM] by electroporation. The use of pUC19-Kan for endonuclease expression required that the acuIM gene be subcloned into pACYC184, a vector with a compatible origin of replication. (see description in section 5). Thirty-six pUC19-Kan colonies were grown to mid-log phase and cell extract was prepared. None of the extracts exhibited AcuI endonuclease activity when incubated with substrate pUC19.
To further address the issue of polymerase fidelity, KOD HiFi polymerase (Novagen (Madison, Wis.) product #71085-3) was used to amplify the acuIRM ORF with primers 291-287 and 291-044. The product was ligated into pUC19-Kan, transformed into ER2744 [pACYC184-AcuIM] and plated on LB-Kan,Cam plates. Thirty-six colonies were grown to mid-log phase and cell extract was prepared. Again, none of the extracts exhibited AcuI endonuclease activity when incubated with lambda DNA. The insert frequency of these thirty-six clones was analyzed by colony PCR and thirty-four of thirty-six clones appeared to contain acuIRM inserts. At this point, a decision was made to employ a third expression vector, pET28a(KanR).
5. Subcloning of acuIM from pUC19 (Clone ANS6) to pACYC184 and Preparation of an AcuI Over-Expression Host.
The acuIM containing insert of clone ANS6 is 2.8 kb. A SalI site is present near one end of the insert upstream from the acuIM start codon. A second SalI site is present within the pUC19 polylinker. Thus, SalI digestion was used to transfer a 2.8 kb acuIM fragment into pACYC184 prepared by SalI digestion and CIP treatment. The in vivo function of acuIM within pACYC184 was verified by digesting plasmid isolates with AcuI endonuclease. Several isolates displayed complete resistance to AcuI digestion and clone #9 was chosen for use in host cell pre-modification. Electrocompetent cells were prepared from clone #9 to create T7 expression host ER2744 [pACYC184-AcuIM].
6. Over-Expression of acuIRM from pET28a
T7 expression vector pET28a (Novagen (Madison, Wis.) product #69864-3) was prepared by NcoI/BamHI digestion followed by CIP treatment. KOD HiFi polymerase was used to PCR-amplify the acuIR gene using primers 291-287 and 291-044. After NcoI/BamHI digestion and gel-purification, the PCR product was ligated into pET28a (KanR). The ligation mix was transformed into ER2744 [pACYC184-AcuIM] by electroporation and plated on LB-Kan,Cam plates. Eighteen colonies were grown, induced for 3 hours with IPTG and cell extract was prepared. Fifteen of eighteen extracts produced a digestion pattern identical to native AcuI. Clone #28 was chosen for further characterization and sequencing. Sequencing results revealed a one-base deletion near the 3′ end of the acuIRM gene as compared to a sequence derived from the original ANS6 clone. Re-evaluation of inverse PCR sequences and the ANS6 sequence led to the conclusion that the original ANS6 sequence had been misinterpreted and the acuIRM stop codon had been predicted incorrectly. As a result, the gene product from clone 28 is altered at the very C-terminus and yet maintains relatively normal specific activity. Since the sequence of clone 28 was otherwise wild-type, this clone was modified to correct the 3′ end of the acuIRM gene sequence.
7. Modification of Clone 28 to Create Wild-Type acuIRM Clone #288
The 3′ end of the acuIRM gene was corrected by transferring a 290 bp fragment from clone ANS6 into clone 28 to create pET28a-AcuIRM clone #288. A unique PmeI site is present 106 bp upstream from the correct acuIRM stop codon. In addition, an EcoRI site is present 184 bp downstream of the stop codon. Therefore, clone 28 and clone ANS6 were digested with PmeI/EcoRI and the appropriate fragments were gel-purified. (Note that the EcoRI site of clone 28 is present in the pET28a polylinker immediately downstream of the BamHI site initially used for cloning the acuIRM gene). The two fragments were ligated and transformed into ER2744 [pACYC184-AcuIM]. Nine colonies were grown to assay for the over-expression of AcuI. The extract of clone #288 exhibited the same level of AcuI activity as clone #28. Clone #288 was sequenced using the T7 terminator primer to verify that the 3′ end was corrected to match the wild-type sequence.
The expression level of clone #288 was estimated by growing 500 mL of cells, inducing for 3 hours with 0.5 mM IPTG and preparing cell extract from the cell pellet. The yield of AcuI was estimated to be 17,400 units per gram of wet cells using lambda DNA as the substrate. The final AcuI over-production strain is E. coli strain ER2744 [pET28a-AcuIRM, pACYC184-AcuIM].
The present invention is further illustrated by the following Example. This Example is provided to aid in the understanding of the invention and is not construed as a limitation thereof.
The references cited above and below are herein incorporated by reference.
EXAMPLE I
Cloning of AcuI Restriction-Modification System in E. coli 
1. Preparation of Genomic DNA
Genomic DNA is prepared from 7.8 g of Acinetobacter calcoaceticus SRW4 (NEB #1449, New England Biolabs strain collection) by the standard procedure consisting of the following steps:
a. Cell lysis by resuspending cells in 50 mM Tris-HCl (pH 8.0), 0.1 M EDTA and addition of lysozyme (2.8 mg/ml final conc.).
b. Further cell lysis by addition of SDS at a final concentration of 1.25%.
c. Further cell lysis by addition of Triton X-100 at a final concentration of 1.0%.
d. Addition of 25 ml TE (pH 8.0) and 40 ml distilled water to improve DNA yield during phenol-chloroform extraction.
e. After freezing overnight at −20° C., proteins are removed by phenol-chloroform extraction four times (equal volume) and chloroform extraction once (equal volume).
f. Dialysis in 4 liters of TE buffer, buffer change twice.
g. RNase A treatment to digest RNA (0.1 mg/ml final conc.).
h. Genomic DNA concentration was estimated to be 0.3 mg/ml so DNA precipitation is not necessary.
i. Genomic DNA yield was estimated to be 6 mg.
2. Restriction Digestion of Genomic DNA and Construction of Genomic DNA Libraries
AcuI genomic DNA (9 μg per reaction) is completely digested with BamHI, EcoRI or SphI or partially digested with ApoI, Sau3AI or NlaIII. The partial digests are optimized to yield DNA fragments in the 2-10 kb range by using varying amounts of ApoI, Sau3AI and NlaIII restriction endonuclease in 30 min reactions. After gel purification of the six fragment types, each lot is ligated into vector pUC19. Vector pUC19 possesses two AcuI (Eco57I) sites, a necessary feature for the methylation selection procedure. The BamHI and Sau3AI fragments are ligated into BamHI cut, CIP-treated pUC19. The EcoRI and ApoI fragments are ligated into EcoRI cut, CIP-treated pUC19. And the SphI and NlaIII fragments are ligated into SphI cut, CIP-treated pUC19. The six ligation reactions are incubated overnight and then each is transformed into ER2502 by electroporation. (ER2502 is strain RR1, endA). Each ligation each yielded >30,000 colonies (except the BamHI ligation yielded only 18,000 colonies). An aliquot of each ligation is separately transformed into ER2683 to conduct blue/white screening to assess the number of insert-containing clones. The percentage of inserts in each library varied from 50-87% (except the BamHI assessment found only 1 of 13 white colonies). Therefore, the BamHI library was excluded. The “complete” library was created by inoculating approximately 10,000 colonies of each of the EcoRI and SphI libraries into 500 mL LB plus Amp, growing for 3 hours and isolating plasmid DNA by the Qiagen (Studio City, Calif.) Maxiprep procedure. The “partial” library was created in the same manner by pooling approximately 10,000 ApoI colonies, 10,000 NlaII colonies and 20,000 Sau3AI colonies.
3. Challenge of AcuI Vector Libraries to Isolate the AcuI Methylase Gene.
Each library (5 μg) is challenged with 20 units of native AcuI overnight at 37° C. in a 250 μL reaction. After heat inactivation of AcuI, small aliquots (1, 2, 5, and 10 μL) are transformed into ER2502 and plated on ampicillin agar plates. Each transformation yielded 5-20 colonies after incubation at 37° C. overnight. Nine colonies from the partial library and nine colonies from the complete library were grown for 6 hrs and plasmid DNA was prepared. The eighteen clones were digested with 4 units of native AcuI to test for the presence of a functional AcuI methylase gene. Three clones were protected from AcuI digestion and thus potentially carried the AcuI methylase gene. These clones designated ANS3, ANS6 and ANS8 were sequenced with pUC19 primers s1233s and s1224s (New England Biolabs (Beverly, Mass.)) to reveal a partial open reading frame with similarity to the eco57IM gene. Clone ANS6 was chosen for further characterization and the 2.8 kb gene insert was confirmed to encode the AcuI methylase gene (acuIM). The vector/insert junction revealed that clone ANS6 was isolated from the NlaIII partial library. Methylase clone ANS6 was completely sequenced by primer walking to define a 1608 bp ORF that encodes a protein of 535 amino acids with high similarity to the amino-methyltransferase family.
4. Cloning of the AcuI Restriction Endonuclease Gene (acuIRM) by Inverse PCR.
The acuIRM gene encoding the AcuI endonuclease-methylase fusion protein resides upstream of the acuIM gene. The AcuI gene organization differs from the Eco57I gene organization (see FIG. 2). Inverse PCR was conducted to characterize the genomic region upstream of acuIM. Inverse PCR primers 287-003 and 287-004 were designed to anneal upstream from the acuIM start codon. Round one inverse PCR primer sequences are as follows:








(SEQ ID NO:5)











5′ tataagctctttttgcttggtcgc 3′
(287-003)







(SEQ ID NO:6)










5′ aagagttctgaccccattgcaacg 3′
(287-004)






AcuI genomic DNA was digested with the following enzymes: AciI, ApoI, BsaAI, BstZ17I, DraI, HincII, NsiI and XmnI. After self-ligation of the digestion products (at 4 ng/μl), inverse PCR was performed using 35 cycles. Inverse PCR products were obtained from the following reactions: ApoI (0.8 kb), HincII (1.8 kb), XmnI (0.8 kb) and AciI (0.4 kb). The ApoI, HincII and XmnI inverse PCR products were gel-purified and sequenced with primers 287-003 and 287-004. The resulting 1.8 kb sequence revealed an incomplete open reading frame with high similarity to the Eco57I endonuclease-methylase fusion gene. This partial ORF was assumed to be acuIRM. As this gene was expected to be approximately 3.0 kb, a second round of inverse PCR was performed.
Round two inverse PCR primer sequences are as follows:








(SEQ ID NO:7)











5′ tacctgttggaattaattgagaag 3′
(288-109)







(SEQ ID NO:8)










5′ tcggtacttataagctgtcttatg 3′
(288-110)






AcuI genomic DNA was digested with the following enzymes: AciI, Sau3AI, Apol, DraI, TseI, SfcI, BclI and BsrBI. After self-ligation of the digestion products (at 4 ng/μl), inverse PCR was performed using 35 cycles. Round two PCR products ranged from 0.6-2.0 kb. A 1.0 kb fragment derived from the DraI digest and a 2.0 kb fragment derived from the TseI digest were gel-purified. Sequencing was carried out using primers 288-109, 288-110 and 288-277.








(SEQ ID NO:9)











5′ cccagagtaaacggactctcttcc 3′
(288-277)






The second round sequence revealed an in-frame GTG codon immediately following a TAA stop codon approximately 3 kb upstream of the acuIM gene. This GTG is the start of the acuIRM gene. Translation can be initiated at this valine codon when other essential genetic elements are present. The acuIRM ORF is 3003 bp, which encodes a protein of 1000 amino acids with a calculated molecular weight of 115,826 daltons.
5. Subcloning of the acuIM Gene into pACYC184
The acuIM containing insert of clone ANS6 is 2.8 kb. A SalI site is present near one end of the insert upstream from the acuIM start codon. A second SalI site is present within the pUC19 polylinker. Thus, SalI digestion was used to transfer a 2.8 kb acuIM fragment into pACYC184 prepared by SalI digestion and CIP treatment. The in vivo function of acuIM within pACYC184 was verified by digesting plasmid isolates with AcuI endonuclease. Several isolates displayed complete resistance to AcuI digestion and clone #9 was chosen for use in host cell pre-modification. The host cells used for AcuI over-expression were ER2744 [pACYC184-AcuIM].
6. Cloning of the 3003 bp ORF Upstream of acuIM to Confirm the Identity of acuIRM
The gene product of the putative acuIRM ORF is 51% identical to the Eco57I restriction endonuclease. The identity and function of the acuIRM gene product was confirmed by cloning the gene into the E. coli T7 expression vector pET28a (KanR). Two PCR primers were synthesized for PCR amplification of the 3003 bp ORF from Acinetobacter calcoaceticus SRW4 genomic DNA:









5′ ccaactgcaggaataacccatggttcatgatcataagcttgaa 3′
(SEQ ID NO:10)



(forward primer 291-287, underline = NcoI site)


5′ ccttccggatccttaatataagggatcaagg 3′
(SEQ ID NO:11)


(reverse primer 291-044, underline = BamHI site)






PCR conditions were 94° C. for 2 min (1 cycle); 95° C. for 15 sec, 55° C. for 30 sec, 72° C. for 60 sec (18 cycles); 72° C. for 7 min (1 cycle). KOD HiFi polymerase (2 units) was used for PCR amplification. The PCR product was purified by Qiagen (Studio City, Calif.) spin column, digested with NcoI and BamHI at 37° C., purified by excision from a low-melt agarose gel and ligated to CIP treated pET28a with compatible ends. Following overnight ligation, the DNA was dialyzed against distilled water for 4 hours and transformed into ER2744 [pACYC184-AcuIM] by electroporation. The transformation mix was plated on LB-agar plus Kan, Cam. Eighteen colonies were grown (in 10 ml LB plus Kan/Cam), induced for 3 hours with 0.5 mM IPTG and cell extract was prepared by sonication. Fifteen of eighteen extracts produced a lambda digestion pattern identical to native AcuI. Clone #28 was chosen for further characterization and sequencing. Sequencing results revealed a one-base deletion near the 3′ end of the acuIRM gene as compared to a sequence derived from the original ANS6 clone. Re-evaluation of inverse PCR sequences and the ANS6 sequence led to the conclusion that the original ANS6 sequence had been misinterpreted and the acuIRM stop codon had been predicted incorrectly. As a result, the gene product from clone 28 is altered at the very C-terminus and yet maintains relatively normal specific activity. Since the sequence of clone 28 was otherwise wild-type, this clone was modified to correct the 3′ end of the acuIRM gene sequence.
7. Modification of Clone 28 to Create Wild-Type acuIRM Clone #288
The 3′ end of the acuIRM gene was corrected by transferring a 290 bp fragment from clone ANS6 into clone 28 to create pET28a-AcuIRM clone #288. A unique PmeI site is present 106 bp upstream from the correct acuIRM stop codon. In addition, an EcoRI site is present 184 bp downstream of the stop codon. Therefore, clone 28 and clone ANS6 were digested with PmeI/EcoRI and the appropriate fragments were gel-purified. (Note that the EcoRI site of clone 28 is present in the pET28a polylinker immediately downstream of the BamHI site initially used for cloning the acuIRM gene). The two fragments were ligated and transformed into ER2744 [pACYC184-AcuIM]. Nine colonies were grown (in 10 ml LB plus Kan/Cam) to assay for the over-expression of AcuI. The extract of clone #288 exhibited the same level of AcuI activity as clone #28. Clone #288 was sequenced using the T7 terminator primer to verify that the 3′ end was corrected to match the wild-type acuIRM sequence.
8. Design of Correct acuIRM Reverse Primer for Subsequent PCR Amplification
Any subsequent manipulation of the acuIRM gene requires a proper reverse PCR primer that anneals downstream of the stop codon. Primer 293-171 (listed below) allows for PCR amplification of the acuIRM gene from pET28a (clone #288) when paired with forward primer 291-287 using the same PCR conditions described in section 6.









5′ ccttccggatccacgtaatttttcggcagatgc 3′
(SEQ ID NO:12)



(reverse primer 293-171, underline = BamHI site)






9. Estimation of AcuI Yield

AcuI yield was estimated by growing clone #288 in LB plus Kan/Cam to late log phase, inducing for 3 hours with 0.5 mM IPTG and preparing cell extract from the cell pellet. The yield of recombinant AcuI was estimated to be 17,400 units per gram of wet cells using lambda DNA as the substrate (see FIG. 5). The AcuI over-production strain E. coli ER2744 [pET28a-AcuIRM, pACYC184-AcuIM] has been deposited under the terms and conditions of the Budapest Treaty with the American Type Culture Collection on Apr. 15, 2003 and received the Accession No. PTA-1513.

## Claims

1. Isolated DNA encoding the AcuI restriction endonuclease, wherein the isolated DNA is obtainable from Acinetobacter calcoaceticus SRW4.

2. A recombinant DNA vector comprising a vector into which a DNA segment encoding the AcuI restriction endonuclease has been inserted.

3. Isolated DNA encoding the AcuI restriction endonuclease and AcuI methylase, wherein the isolated DNA is obtainable from ATCC No. PTA-1513.

4. A cloning vector that comprise the isolated DNA of claim 3.

5. A host cell transformed by the vector of claim 2 or 4.

6. A method of producing recombinant AcuI restriction endonuclease comprising culturing the host cell of claim 5 under conditions suitable for expression of said endonuclease and methylase.