METHOD TO PRODUCE ENZYMATICALLY ACTIVE RECOMBINANT

ENDONUCLEASES

This patent application claims the benefit of priority from U.S. Provisional Application Serial No. 60/776,770, filed February 24, 2006, teachings of which are herein incorporated by reference in their entirety.

Background of the Invention

A novel family of DNA mismatch-specific endonucleases from plants was discovered recently (Oleykowski, et al .

(1998) Nucl. Acid Res. 26:4597-4602; Yang, et al . (2000)

Biochem. 39:3533-3541). The plant source with the highest apparent concentration of this class of endonucleases is celery (Oleykowski, et al . (1998) supra), and thus the enzyme was purified from celery and named CEL I (Oleykowski, et al . (1998) supra; Yang, et al . (2000) supra) . CEL I cleaves DNA at the 3' -side of sites of base- substitution mismatches and DNA distortion (Oleykowski, et al. (1998) supra; Yang, et al . (2000) supra). CEL I has been shown to be useful in mismatch detection assays that rely on nicking or cleaving duplex

DNA at insertion/deletion and base-substitution mismatches

(Oleykowski, et al . (1998) supra; Yang, et al . (2000) supra; Kulinski, et al . (2000) BioTechniques 29:44-48; Colbert, et al . (20001) Plant Physiol. 126:480-484; Sokurenko, et al . (2001) Nucl. Acids Res. 29:elll; U.S. Patent No. 5,869,245).

Purified preparations of CEL nuclease identified as CEL I contain two different protein species, CEL I and CEL II (Yang, et al . (2000) supra; U.S. Patent no. 5,869,245). One species, called CEL I, has an apparent molecular weight of 43 kDa as determined by SDS-PAGE. Removal of N-linked oligosaccharides with Endo H _f reduces the molecular weight

to 29 kDa . CEL I was partially sequenced and the gene encoding CEL I was isolated from a celery cDNA library, sequenced, and cloned into E. coli (Yang, et al . (2000) supra; U.S. Patent 5,869,245). CEL II has an apparent molecular weight of 39 kDa as determined by SDS-PAGE and removal of N-linked oligosaccharides reduces the molecular weight to 37 kDa. Chromatographic separation of CEL I and CEL II has been described and while CEL I and CEL II appear to be related, they have different enzymatic activity; CEL II has a higher pH optimum than CEL I and CEL II is more efficient than CEL I in cleaving DNA at mismatches.

The complete gene of CEL II has also been cloned and its DNA sequence determined (WO 2006/007124 published January 19, 2006) . Expression of recombinant CEL I in the yeast strain Pichia pastoris is disclosed in published PCT Application WO 2004/035771. A crude preparation of the recombinant CEL I produced therein was reported to nick DNA at a site next to all 8 possible mismatches. However, nothing more was reported about the enzymatic properties of the cloned enzyme .

Published U.S. Patent Application No. 2003/0157682 describes expression of recombinant CEL I using a GENEWARE Cloning system in tobacco leaves. However, the expressed recombinant CEL I exhibits more nonspecific nuclease activity that native CEL I and is not suitable for commercialization.

No published reports have been observed to date describing expression of recombinant enzymatically active CEL I or CEL II protein in bacterial, mammalian or insect cells .

There is a need for recombinant methods for production of functionally active plant DNA endonucleases such as CEL I and CEL II.

Summary of the Invention

The present invention provides a method for recombinant expression of a functionally active plant DNA endonuclease which comprises culturing cells capable of recombinantly expressing a plant DNA endonuclease in a cell culture media supplemented with Zn ⁺⁺ .

Brief Description of the Figures

Figure 1 shows clonal selection of a stable 293T cell line expressing recombinant CEL II. In this photograph of an electrophoresis gel, Lane M is a 100 bp DNA size marker; Lanes B2 , B4 , B6, B8, B9 and BlO are geneticin resistant clones, with B2 , B4 , B6, B8 and B9 expressing CEL II activity as assayed in a Control G/C mismatch cutting assay; Lane P is a negative control with supernatant from vector-transfected cells; and Lane S is 10 units of SURVEYOR Nuclease S. Both B6 and B9 were further expanded. After expansion, B6 was selected for further work because it had higher mismatch cutting activity than B9 post expansion. Figure 2 shows an assay of CEL II mismatch cutting activity in the culture supernatant of the B-6 clone of human 293 cells. Mismatch cutting activity in the culture supernatant of the B-6 clone of 293 cells grown in 15-cm dishes was assayed with Control G/C heteroduplex analyzed on a 1.5% agarose gel. In this photograph of an electrophoresis gel, Lane M is a 100 bp DNA size marker; Lane rCEL2 shows activity of recombinant CEL II expressed

by B- 6 clone; and Lane SNS is 10 units of SURVEYOR Nuclease .

Figure 3 shows an assay of CEL II mismatch cutting activity in the culture supernatant of the B- 6 clone of human 293 cells grown in spinner suspension culture. In this photograph of an electrophoresis gel, Lane M is a 100 bp DNA size marker; Lane U is uncut Control G/C; Lane Sp is Control G/C treated with recombinant CEL II recovered from B-6 cell spinner culture maintained in 293 SFMII + 20 μM ZnCl ₂ ; and Lane S is 10 units of SURVEYOR Nuclease.

Figure 4 shows an assay of CEL II mismatch cutting activity in the culture supernatant of the B-6 Clone of human 293 cells grown through two expansions in spinner suspension culture. In this photograph of an electrophoresis gel, Lane M is a 100 bp DNA size marker; Lane U is uncut Control G/C; Lane Sp is Control G/C treated with recombinant CEL II recovered from B-6 cell spinner culture maintained in 293 SFMII + 20 μM ZnCl ₂ through two rounds of growth; and Lane S is 10 units of SURVEYOR Nuclease.

Detailed Description of the Invention

Members of the CEL nuclease family of plant DNA endonucleases have the unique ability to cut DNA heteroduplexes at mismatches at neutral and alkaline pH. CEL I and CEL II have been used extensively in mutation detection in genetic variant discovery and diagnosis of genetic disease.

Based on the distinct enzymatic activities of CEL I and CEL II and their utility in assays for detecting the presence of mismatches in DNA, it is desirable to produce highly pure preparations of CEL I and CEL II on a large scale .

Currently the commercial production of CEL I and/or CEL II is based on extraction and purification of native enzyme from celery stalks. However, recombinant production which is not subject to variations of CEL I or CEL II levels related to celery strain and growth conditions would be preferred. Further, the ability to affinity tag the recombinantly produced endonuclease would greatly simplify purification for production, would eliminate contamination by extraneous plant nucleases that tend to co-purify with native enzyme, and would make production more reproducible and economical .

However, while the complete genes of both CEL I and CEL II have been cloned and their DNA sequences determined, previous attempts by the inventors to express recombinant enzymatically active protein in bacterial, mammalian or insect cells have been unsuccessful . Extensive efforts by the inventors to date to express enzymatically active recombinant CEL I and CEL II in E. coli have been unsuccessful . Attempts to denature and renature an insoluble bacterial protein failed to reconstitute mismatch cutting activity. Folding of recombinant CEL II in the E. coli periplasmic space through the inclusion of appropriate amino-terminal leader sequences and expression in redox- modified host strains (available from Novagen) also failed to produce active protein. Engineering of CEL I to replace four or five of eight cysteines with other amino acids did not produce active protein in E. coli. Transfection of cultured human 293 cells with CEL II gene constructs failed to produce detectable levels of DNA mismatch cutting activity in or outside cells. Attempts to establish a permanent insect cell line using a baculovirus expression system to produce active CEL I were also unsuccessful .

CEL I and CEL II are homologues of the Pl/Sl nuclease family. Critical catalytic and structural amino acids are highly conserved across family members including CEL I and CEL II. Based upon extrapolation of Pl nuclease biochemical and three-dimensional structural data (Volbeta et al. (1991) EMBO J 10:1607-1618) to CEL I and CEL II, these enzymes bind three Zn ⁺⁺ atoms that are required for catalysis. Removal of Zn ⁺⁺ from Pl nuclease with EDTA causes irreversible inactivation of the enzyme. It has now been found that Zn ⁺⁺ is also critical to proper protein folding during protein processing and maturation. The amount of zinc in most culture media is not sufficient for proper folding of a nascent peptide and structural stabilization. As demonstrated herein, supplementing the cell culture media of a recombinant expression system for a plant DNA endonuclease such as CEL I or CEL II results in expression of a functionally active enzyme. In these experiments, addition of 10 μM ZnCl ₂ to the culture medium of human 293 cells bearing a cloned CEL II gene resulted in production and secretion of a CEL II protein with DNA heteroduplex mismatch cutting activity in the cell culture supernatant. While the exemplary experiments described herein are directed to recombinant expression of CEL II, it is expected based upon similarities in this enzyme family that supplementing the cell culture media with Zn ⁺⁺ will also provide a useful method for recombinant expression of CEL I as well as other plant DNA endonucleases, particularly Zn ⁺⁺ -dependent enzymes that are currently only available in non-recombinant forms, e.g. mung bean, Pl and Sl nuclease.

Accordingly, the present invention provides a method for recombinant expression of functionally active plant DNA endonucleases such as functionally active CEL I or

functionally active CEL II wherein Zn ⁺⁺ is included in the cell culture media. In a preferred embodiment, Zn ⁺⁺ is added to the cell culture media as a soluble salt of Zn ²⁺ such as, but not limited to, ZnCl ₂ , Zn(CH ₃ COO) ₂ , or ZnSO ₄ , at a concentration ranging from about 1 μM to about 100 μM, preferably about 10 μM. Experiments described herein used DMEM cell culture media from GIBCO (Invitrogen) supplemented with Zn ⁺⁺ . However, it is expected that any cell culture media used routinely by those skilled in the art in recombinant methods can be supplemented with Zn ⁺⁺ for use in the method of the present invention.

As used herein, a functionally active endonuclease is a recombinantly expressed endonuclease that retains at least one biological activity associated with the native endonuclease. By retains biological activity, it is meant that the recombinantly expressed plant DNA endonuclease retains at least about 10%, 20%, 30%, 40%, 50%, 60%, 75%, 85%, 90%, 95%, 97%, 98%, 99%, or more, of the biological activity of the native endonuclease and can even have a higher level of activity than the native endonuclease.

In a preferred embodiment, the endonuclease expressed recombinantly in the method of the present invention is a plant DNA endonuclease, more preferably a Zn ⁺⁺ -dependent enzyme such as mung bean, Pl and Sl nuclease or a CEL I or II protein, even more preferably a CEL I or CEL II protein.

By CEL I protein or CEL II protein as used herein, it is meant to encompass an enzyme capable of cleaving double- stranded DNA at base pair mismatches. The terms CEL I protein and CEL II protein also include modified (e.g., mutated) CEL I or CEL II proteins that retain biological function (i.e., have at least one biological activity of the native CEL I protein or CEL II protein, e.g., cleaving

double-stranded DNA at base pair mismatches or binding double-stranded DNA at base pair mismatches) , functional CEL I or II fragments including, but not limited to, truncated molecules and functional CEL I or CEL II fusion polypeptides (e.g., a CEL I-GST or CEL II-GST fusion protein or a CEL I -His or CEL II -His tagged protein) .

RecombinantIy expressed CEL I and CEL II proteins can be produced from cells expressing CEL I or CEL II nucleic acid sequences, the sequences being derived not only from celery, but also from other plant species including, but not limited to, Arabidopsis, Zinnia, alfalfa, asparagus, tomato, cauliflower, broccoli, cabbage, fennel, kale, water cress, parsley, lettuce, Swiss chard, onion, mung bean sprouts, Hemerocallis, Oryza, Hordeum, or Zea. Exemplary cDNA sequences of a celery CEL II useful in the methods of the present invention are disclosed in PCT Application No. PCT/US2005/017508, teachings of which are herein incorporated by reference in their entirety, as well as in GENBANK accession numbers AAD00695, BAB03377, AAC34856, CAC33831 and NP_680734. An exemplary cDNA sequence of CEL I useful in the methods of the present invention is disclosed in GENBANK accession number AF237958. Additional nucleic acids for use in recombinant expression of CEL I or CEL II in accordance with the methods of the present invention include, but are not limited to those having at least about 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98% or higher sequence similarity with the isolated nucleic acid sequences disclosed supra (or fragments thereof, as defined above) or those encoding a CEL I or CEL II polypeptide having at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98% or higher amino acid sequence identity with a polypeptide sequence disclosed in PCT Application No. PCT/US2005/017508 or GENBANK accession number AAD00695,

BAB03377, AAC34856, CAC33831, NP_680734, or AF237958 (or fragments thereof) , teachings of which are incorporated herein by reference, and which produce upon recombinant expression a functionally active CEL I or CEL II protein as defined herein.

The ability to express active recombinant endonucleases such as CEL I or CEL II using the method of the present invention also allows for alteration of targeted amino acid residues to reduce intrinsic exonuclease and non-specific endonuclease activity and to enhance mismatch cutting specificity. Further, the catalytic domain of the recombinantly expressed endonuclease can be modified so that the expressed protein retains the ability to bind a mismatch substrate without, for example, cleaving the substrate. Such modified proteins can be used to increase the sensitivity of mismatch detection by increasing the proportion of heteroduplex in a DNA population. Tagged or labeled recombinantly expressed endonucleases with binding activity can also be used in in situ mutation staining or affinity based mutation detection methods.

To modify endonuclease amino acid sequences specifically disclosed herein or otherwise known in the art, amino acid substitutions can be based on any characteristic known in the art, including the relative similarity or differences of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. In particular embodiments, conservative substitutions (i.e., substitution with an amino acid residue having similar properties) are made in the amino acid sequence encoding, for example CEL I or CEL II. Such modifications can be made to alter the pH

optimum, temperature optimum or stability of CEL I or CEL II .

In making amino acid substitutions, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (see, Kyte and Doolittle (1982) J. MoI. Biol. 157:105) . It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.

Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte and Doolittle (1982) supra), and these are: isoleucine (+4.5); valine (+4.2); leucine

(+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

It is also understood in the art that the substitution of amino acids can be made on the basis of hydrophilicity . U.S. Patent 4,554,101 states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein. As detailed in U.S. Patent 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (±3.0); aspartate (+3.0 ± 1); glutamate (+3.0 ± 1); serine (+0.3); asparagine (+0.2);

glutamine (+0.2); glycine (0); threonine (-0.4); proline (- 0.5 ± 1); alanine (-0.5); histidine (-0.5); cysteine (- 1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).

The isolated nucleic acid sequence encoding the endonuclease is preferably associated with an expression control sequence, e.g., a transcription/translation control signals and/or a polyadenylation signal. It will be appreciated that a variety of promoter/enhancer elements can be used depending on the level and cell- -specific expression desired. The promoter can be constitutive or inducible (e.g., the metallothionein promoter or a hormone inducible promoter) , depending on the pattern of expression desired. The promoter can be native or foreign and can be a natural or a synthetic sequence. By foreign, it is intended that the transcriptional initiation region is not found in the wild-type host into which the transcriptional initiation region is introduced. The promoter is chosen so that it will function in the target cell (s) of interest. In particular embodiments, the promoter functions in cells that can be used to recombinantly express nucleic acids encoding endonucleases for the purposes of large-scale protein production. Likewise, the promoter can be specific for these cells

(i.e., only show significant activity in the specific cell type) .

For example, a CEL II coding sequence can be operatively associated with a cytomegalovirus (CMV) major immediate-early promoter, an albumin promoter, an

Elongation Factor 1-α (EFl-α) promoter, a PγK promoter, a MFG promoter, a Rous sarcoma virus promoter, or a glyceraldehyde-3 -phosphate promoter .

Moreover, specific initiation signals are generally required for efficient translation of inserted protein coding sequences. These translational control sequences, which can include the ATG initiation codon and adjacent sequences, can be of a variety of origins, both natural and synthetic. For example, it can be appreciated by one of skill in the art that CEL II lacking the endogenous signal peptide would require the addition of an initiation codon at the 5' end of the coding sequence for recombinant expression of the mature CEL II protein.

Endonucleases can be recombinantIy expressed not only directly, but also as a fusion protein with a heterologous polypeptide, i.e. a signal sequence for secretion and/or other polypeptide which will aid in the purification of the endonuclease . In one embodiment, the heterologous polypeptide has a specific cleavage site to remove the heterologous polypeptide from the endonuclease.

In general, a signal sequence can be an endogenous signal sequence or a component of the vector and should be one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. For production in a prokaryote, a prokaryotic signal sequence from, for example, alkaline phosphatase, penicillinase, lpp, or heat- stable enterotoxin II leaders can be used. For yeast secretion, one can use, e.g., the yeast invertase, alpha factor, or acid phosphatase leaders, the Candida albicans glucoamylase leader (EP 362,179), or the like (see, for example WO 90/13646) . In mammalian cell expression, signal sequences from secreted polypeptides of the same or related species, as well as viral secretory leaders, for example, the herpes simplex glycoprotein D signal can be used. Such signal sequences can advantageously be fused to nucleic acid sequences coding for the mature endonuclease.

Other useful heterologous polypeptides which can be fused to the endonuclease include, but are not limited to, those which increase expression or solubility of the fusion protein or aid in the purification of the fusion protein by acting as a ligand in affinity purification. Typical fusion expression vectors include pGEX (Amersham Biosciences, Piscataway, NJ), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, NJ) which fuse GST, maltose E binding protein or protein A, respectively, to the target recombinant protein.

In the method of the present invention, the isolated nucleic acid sequence encoding the endonuclease is incorporated into a vector for recombinant protein production. Expression vectors compatible with various host cells are well-known in the art and contain suitable elements for transcription and translation of nucleic acids. Typically, an expression vector contains an expression cassette, which includes, in the 5' to 3 ' direction, a promoter, a coding sequence encoding the endonuclease operatively associated with the promoter, and, optionally, a termination sequence including a stop signal for RNA polymerase and a polyadenylation signal for polyadenylase . Exemplary vectors include, but are not limited to, bacterial artificial chromosomes, cosmids, yeast artificial chromosomes, phage, plasmids, lipid vectors, poly-lysine vectors, synthetic polyamino polymer vectors and viral vectors (e.g., retrovirus, alphavirus, vaccinia virus, adenovirus, adeno-associated virus, or herpes simplex virus) .

As used herein, the term viral vector or viral delivery vector can refer to a virus particle that functions as a nucleic acid delivery vehicle, and which

contains the vector genome packaged within a virion. Alternatively, these terms can be used to refer to the vector genome when used as a nucleic acid delivery vehicle in the absence of the virion. Protocols for producing recombinant viral vectors and for using viral vectors for nucleic acid delivery can be found in Current Protocols in Molecular Biology, Ausubel, F. M. et al . (eds.) Greene Publishing Associates, (1989) and other standard laboratory manuals (e.g., Vectors for Gene Therapy. In: Current Protocols in Human Genetics . John Wiley and Sons, Inc. : 1997) .

Expression vectors can be designed for expression of polypeptides in prokaryotic or eukaryotic cells. For example, polypeptides can be expressed in bacterial cells such as E. coli, insect cells (e.g., in the baculovirus expression system), yeast cells or mammalian cells. Some suitable host cells are discussed further in Goeddel (1990) Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA. Examples of vectors for expression in yeast S. cerevisiae include pYepSecl (Baldari, et al . (1987) EMBO J. 6:229-234), pMFa (Kurjan and Herskowitz (1982) Cell 30:933-943), pJRY88 (Schultz, et al. (1987) Gene 54:113-123), and pYES2 (INVITROGEN Corporation, San Diego, CA) . Baculovirus vectors available for expression of nucleic acids to produce proteins in cultured insect cells (e.g., Sf 9 cells) include the pAc series (Smith, et al . (1983) MoI. Cell. Biol. 3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170:31-39) . Examples of mammalian expression vectors include pCDM8 (Seed (1987) Nature 329:840) and pMT2PC (Kaufman, et al . (1987) EMBO J. 6:187-195). When used in mammalian cells, the expression vector's control functions are often

provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus and Simian Virus 40.

In addition to the regulatory control sequences discussed herein, the recombinant expression vector can contain additional nucleotide sequences. For example, the recombinant expression vector can encode a selectable marker gene to identify host cells that have incorporated the vector. Vectors can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms transformation and transfection refer to a variety of art- recognized techniques for introducing foreign nucleic acids (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, microinjection, DNA-loaded liposomes, lipofectamine-DNA complexes, cell sonication, λgrojbacteriurn-mediated transformation, gene bombardment using high velocity microprojectiles, and viral -mediated transfection. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al . (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press (1989) ) , and other laboratory manuals.

Often only a small fraction of cells (in particular, mammalian cells) integrate the foreign DNA into their genome. In order to identify and select these integrants, a nucleic acid that encodes a selectable marker (e.g., resistance to antibiotics) can be introduced into the host cells along with the nucleic acid of interest. In particular embodiments, selectable markers include those that confer resistance to drugs, such as G418, hygromycin

and methotrexate. Nucleic acids encoding a selectable marker can be introduced into a host cell on the same vector as that comprising the nucleic acid of interest or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die) .

The recombinant expression methods of the present invention provide a means for expressing nucleic acids encoding a functionally active endonuclease in a broad range of host cells, including both dividing and non- dividing cells in vitro {e.g., for large-scale recombinant protein production) . The nucleic acid can be expressed transiently in the target cell or the nucleic acid can be stably incorporated into the target cell, for example, by integration into the genome of the cell or by persistent expression from stably maintained episomes (e.g., derived from Epstein Barr Virus) . The cell can be a bacterial, protozoan, plant, yeast, fungus, or animal cell.

In particular embodiments, an isolated nucleic acid encoding CEL II can be introduced into a cultured cell, e.g., a cell of a primary or immortalized cell line for recombinant protein production. The recombinant cells can be used to produce the CEL II polypeptide, which is collected from the cells or cell culture medium.

For large-scale production, the recombinantly expressed endonuclease is purified from cultured cells. Typically, the endonuclease is recovered from the culture medium as a secreted polypeptide, although it also can be recovered from host cell lysates when directly expressed without a secretory signal . When the endonuclease is expressed in a recombinant cell other than one of native

origin, it is completely free of proteins or polypeptides of native origin. However, it is necessary to purify the endonuclease from recombinant cell proteins or polypeptides to obtain preparations that are substantially homogeneous as to the recombinant endonuclease. As a first step, the culture medium or lysate is centrifuged to remove particulate cell debris. The membrane and soluble protein fractions are then separated. The recombinant endonuclease can then be purified from the soluble protein fraction. The recombinant endonuclease thereafter can then be purified from contaminant soluble proteins and polypeptides as disclosed herein or with, for example, the following suitable purification procedures: by fractionation on immunoaffinity or ion-exchange columns; ethanol precipitation; reverse phase HPLC; chromatography on silica or on a cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, for example, SEPHADEX™ G-75; and ligand affinity chromatography. Suitably pure preparations of the recombinant endonuclease can be analyzed for functional activity using one or more well-known assays including, but not limited to, a DNA nicking assay, a DNase solubilization assay, or a mismatch endonuclease assay. Purified products can also be analyzed by electrophoretic separation, such as polyacrylamide gel electrophoresis.

Purified CEL I and CEL II preparations produced recombinantly in accordance with the present invention can be used in methods for detecting the presence of mismatches in double-stranded DNA or determining the site of a mutation in double-stranded DNA mismatches in double- stranded DNA. Examples of such methods are found in U.S. Patents 5,869,245 and 6,027,898.

The ability to express endonucleases such as CEL I and CEL II recombinantIy via methods of the present invention also now provides a means to conduct structure- function studies of endonucleases such as CEL II. In these studies, site-directed mutagenesis, deletion/truncation, domain fusion, and related techniques can be used to elucidate the mechanism of mismatch recognition and cutting.

Further the methods of the present invention can be used to express additional active recombinant CEL family members and related enzymes from various plants. The genes of family orthologues can be cloned from cDNA by low stringency hybridization or PCR with degenerate primers and then expressed with the Zn ⁺⁺ supplemented recombinant expression method of the present invention. A mixture of several endonuclease orthologues may provide better mismatch cutting properties, e.g. more normalized mismatch base cutting preferences. Shuffling of domains of endonuclease orthologues of, for example CEL II, to generate novel artificial enzymes of better mismatch cutting properties is also possible.

Accordingly, the ability to recombinantly express functionally active endonucleases such as CEL I and CEL II via the Zn ⁺⁺ supplemented method of the present invention provides significant advantages over known methods of isolation and purification of such enzymes from natural extracts. In particular, the ability to produce functionally active endonucleases such as CEL I and CEL II via recombinant expression makes available consistent production starting material that is not subject to the variations that impact the celery plant or limited in amount due to limited availability of the source. Further, methods of the present invention provide for simplified, more cost effective production, particularly in embodiment

wherein an affinity tag is incorporated into the recombinant protein. The recombinant method of production of the present invention also eliminates contamination by other plant nucleases and makes possible genetic engineering of endonucleases such as CEL I and/or CEL II with improved enzymatic properties and/or broad binding specificity. Thus, the Zn++ supplemented recombinant expression method of the present invention provides an economic means for production of functionally active endonucleases such as CEL I and CEL II which exhibit better quality, less variability, and enhanced and more robust performance of engineered endonucleases.

The following non-limiting examples are provided to further illustrate the present invention.

EXAMPLES

Example 1: Construction of pcDNA3.1/CEL2 and pcDNA3.1/CEL2-

Flag for Expression of CEL II in Cultured Human 293 Cells

The expression vector used to construct pcDNA3.1/CEL2 was pcDNA3. lD/V5-His~TOPO (Invitrogen) . Expression is driven by a CMV promoter and transcription uses a bovine growth hormone polyadenylation site. pcDNA3.1/CEL2 is an expression construct containing a native CEL II open reading frame (ORF) . A STOP codon was included at the 3' end of the ORF. The sequence upstream of the start codon of the ORF was 5 ' -CCTTCACC-3 ' (SEQ ID N0:l) .

The native signal peptide of the CEL II gene was predicted to be functional in human 293T cells by SignalP. The CEL II ORF sequences were PCR amplified with Optimase Polymerase (Transgenomic, Inc.) using 5'- CACCATGGGTATGTTGACTTATACTGG-3' (SEQ ID NO : 2 ) and 5'- TTACACTATTTCAATATTGTTACTGCTTC-3' (SEQ ID NO : 3 ) as the

forward and the reverse primers, the PCR product was ligated to the vector, and the construct was used to transform E. coli competent cells. Plasmid DNA was purified from a culture derived from a transformed colony with a QIAGEN DNA purification column. DNA sequencing of the CEL II gene insert showed the sequence to be 100% correct. The insert region sequence was :

5 ' -CCCTTCACCATGGGTATGTTGACTTATACTGGAATTTATTTTCTGCTATTACTTCC AAGTGTTTTCTGTTGGGGAAAACAAGGACATTTTGCAATTTGTAAAATTGCCCAGGGGT TCCTTAGTAAAGATGCACTGACTGCAGTGAAAGCATTGCTCCCAGAATATGCAGATGGT GATCTAGCAGCTGTTTGCTCCTGGGCTGACGAGGTTCGATTTCATATGCGTTGGAGTAG CCCATTACATTATGTGGACACGCCTGATTTCAGGTGTAACTATAAATACTGTAGAGATT GCCATGATTCTGTTGGACGGAAAGACCGGTGTGTTACTGGAGCAATTCACAACTACACA GAGCAACTTCTATTGGGTGTTCATGACTTGAATTCAAAAATGAATAACAACTTGACGGA GGCACTTATGTTCTTATCACATTTCGTTGGTGATGTCCATCAGCCTCTACATGTTGGCT TCCTTGGCGATGAAGGAGGAAACACAATCACCGTCCGCTGGTATCGGAGGAAAACCAAT TTGCATCATGTATGGGACACAATGATGATTGAATCCTCCTTGAAGACATTCTACAATTC AGATCTTTCTAGCTTAATACAAGCTATTCAGAGCAATATTACAGGTGTCTGGCTTACCG ACAGCTTATCTTGGAGCAATTGCACTGCTGATCATGTGGTTTGTCCAGACCCGTATGCT TCTGAAAGCATTGAGTTGGCCTGCAAGTTTGCCTACAGAAATGCCACACCTGGGACCAC

TTTAGGAGATGAGTACTTCCTCTCTCGGTTGCCTGTTGCGGAGAAGAGGTTGGCTCA GG CTGGGGTCCGTTTGGCTGCTACTCTTAACCGAATCTTCACTTCAAACCCCAGCGATCTC ACAAGATTGAATATGCATAATGGTGGACATAGAAGCAGTAACAATATTGAAATAGTGTA A-3' (SEQ ID NO:4)

Protein sequence:

MGMLTYTGIYFLLLLPSVFCWGKQGHFAICKIAQGFLSKDALTAVKALLPEYADGDL AA

VCSWADEVRFHMRWSSPLHYVDTPDFRCNYKYCRDCHDSVGRKDRCVTGAIHNYTEQ LL

LGVHDLNSKMNNNLTEALMFLSHFVGDVHQPLHVGFLGDEGGNTITVRWYRRKTNLH HV WDTMMIESSLKTFYNSDLSSLIQAIQSNITGVWLTDSLSWSNCTADHWCPDPYASESI

ELACKFAYRNATPGTTLGDEYFLSRLPVAEKRLAQAGVRLAATLNRIFTSNPSDLTR LN MHNGGHRSSNNIEIV (SEQ ID NO: 5) .

Underlining above depicts the signal peptide. pcDNA3.1/CEL2 -Flag is an expression construct containing a native CEL II ORF in frame with a Flag tag at the C-terminus. The CEL II ORF sequences were PCR amplified with Optimase Polymerase using 5'- CACCATGGGTATGTTGACTTATACTGG-S' (SEQ ID NO: 2) and 5'-

TTATTTATCATCATCATCTTTATAATCCACTATTTCAATATTGTTACTGCTTC-3 ' (SEQ ID NO: 6) as the forward and reverse primers, the PCR

product was ligated to the vector, and the construct was used to transform E. coli competent cells. Plasmid DNA was purified from a culture derived from a transformed colony with a QIAGEN DNA purification column. DNA sequencing of the CEL II gene insert showed the sequence to be 100% correct . The insert region sequence was :

5 ' -CACCATGGGTATGTTGACTTATACTGGAATTTATTTTCTGCTATTACTTCCAAGTG TTTTCTGTTGGGGAAAACAAGGACATTTTGCAATTTGTAAAATTGCCCAGGGGTTCCTT AGTAAAGATGCACTGACTGCAGTGAAAGCATTGCTCCCAGAATATGCAGATGGTGATCT AGCAGCTGTTTGCTCCTGGGCTGACGAGGTTCGATTTCATATGCGTTGGAGTAGCCCAT TACATTATGTGGACACGCCTGATTTCAGGTGTAACTATAAATACTGTAGAGATTGCCAT GATTCTGTTGGACGGAAAGACCGGTGTGTTACTGGAGCAATTCACAACTACACAGAGCA ACTTCTATTGGGTGTTCATGACTTGAATTCAAAAATGAATAACAACTTGACGGAGGCAC TTATGTTCTTATCACATTTCGTTGGTGATGTCCATCAGCCTCTACATGTTGGCTTCCTT GGCGATGAAGGAGGAAACACAATCACCGTCCGCTGGTATCGGAGGAAAACCAATTTGCA TCATGTATGGGACACAATGATGATTGAATCCTCCTTGAAGACATTCTACAATTCAGATC TTTCTAGCTTAATACAAGCTATTCAGAGCAATATTACAGGTGTCTGGCTTACCGACAGC TTATCTTGGAGCAATTGCACTGCTGATCATGTGGTTTGTCCAGACCCGTATGCTTCTGA AAGCATTGAGTTGGCCTGCAAGTTTGCCTACAGAAATGCCACACCTGGGACCACTTTAG GAGATGAGTACTTCCTCTCTCGGTTGCCTGTTGCGGAGAAGAGGTTGGCTCAGGCTGGG

GTCCGTTTGGCTGCTACTCTTAACCGAATCTTCACTTCAAACCCCAGCGATCTCACA AG ATTGAATATGCATAATGGTGGACATAGAAGCAGTAACAATATTGAAATAGTGGATTATA AAGATGATGATGATAAATAA-3' (SEQ ID NO: 7) Protein sequence:

MGMLTYTGIYFLLLLPSVFCWGKQGHFAICKIAQGFLSKDALTAVKALLPEYADGDL AA VCSWADEVRFHMRWSSPLHYVDTPDFRCNYKYCRDCHDSVGRKDRCVTGAIHNYTEQLL LGVHDLNSKMNNNLTEALMFLSHFVGDVHQPLHVGFLGDEGGNTITVRWYRRKTNLHHV WDTMMIESSLKTFYNSDLSSLIQAIQSNITGVWLTDSLSWSNCTADHWCPDPYASESI

ELACKFAYRNATPGTTLGDEYFLSRLPVAEKRLAQAGVRLAATLNRIFTSNPSDLTR LN MHNGGHRSSNNIEIVDYKDDDDK (SEQ ID NO: 8) .

The underlining above indicates the signal peptide and the bolding indicates the flag tag.

Example 2 : Transient Expression of CEL II in Cultured Human 293 Cells

The plasmid DNAs were transiently transfected into 293T cells with Lipofectamine (Invitrogen) in 10-cm culture dishes. pUC19 plasmid DNA was used as mock transfection control .

Aliquots (5, 25, 50 μL) of culture supernatants at 3- days post-transfection, as well as cell lysates, were assayed for single-stranded DNA solubilization activity. A slight increase in nuclease activity over mock- infected cells was observed (approximately a 1.6 fold increase) .

Single-stranded DNA solubilization activity was assayed in a 50-μL reaction mixture containing 20 mM Tris- HCl (pH 8.5), 10 mM KCl, 3 mM MgCl ₂ , 0.5 mg/ml denatured calf thymus DNA, and CEL II nuclease incubated at 37 ⁰ C for 10 minutes. The amount of digested DNA was determined by measuring A ₂₆ o in a spectrophotometer after precipitating undigested DNA with acid. One unit of solubilization activity is the amount of enzyme required to produce 1 ng of acid-soluble material in 1 minute at 37°C. No mismatch cutting activity with Control G/C DNA substrate containing G/G and C/C mismatches was observed in culture supernatants or cell lysates from cells transfected with either CEL II or CEL II-flag constructs.

The mismatch cutting assay was performed as follows. Two plasmid DNAs, Control G and Control C, were amplified by PCR with Optimase Polymerase. These two control DNAs are plasmids with inserts that differ at a single base pair. The sequence of the PCR product for Control G is shown below. Control C differs from Control G because it has a C in lieu of the G (underlined) . Primer sequences are underlined at the 5' and 3' end of the amplicon sequence.

ACACCTGATCAAGCCTGTTCATTTGATTACCAGAGAGACTGTCATGATCCACATGGA GG GAAGGACATGTGTGTTGCTGGAGCCATTCAAAATTTCACATCTCAGCTTGGCCATTTCC GCCATGGAACATCTGATCGTCGATATAATATGACAGAGGCTTTGTTATTTTTATCCCAC TTCATGGGAGATATTCATCAGCCTATGCATGTTGGATTTACAAGTGATATGGGAGGAAA

CAGTATAGATTTGCGCTGGTTTCGCCACAAATCCAACCTGCACCATGTTTGGGATAG AG AGATTATTCTTACAGCTGCAGCAGATTACCATGGTAAGGATATGCACTCTCTCCTACAA GACATACAGAGGAACTTTACAGAGGGTAGTTGGTTGCAAGATGTTGAATCCTGGAAGGA ATGTGATGATATCTCTACTAGCGCCAATAAGTATGCTAAGGAGAGTATAAAACTAGCCT GTAACTGGGGTTACAAAGATGTTGAATCTGGCGAAACTCTGTCAGATAAATACTTCAAC

ACAAGAATGCCAATTGTCATGAAACGGATAGCTCAGGGTGGAATCCGTTTATCCATG AT TTTGAACCGAGTTCTTGGAAGCTCCGCAGATCATTCTTTGGCG (SEQ ID NO: 9)

PCR amplification of 10 ng of each DNA in a 50 -μL PCR reaction produces >25 ng/μL of a 632 -bp PCR product. Control G- and Control C-amplified DNA in equal amounts (15 μL of each at >40 ng/μL) are hybridized directly in PCR buffer. This produces a population of molecules containing 50% homoduplex, 25% heteroduplex with a C/C mismatch, and 25% heteroduplex with a G/G mismatch.

Reaction mixtures (20 μL) containing IX Optimase PCR Reaction Buffer, 200 ng Control G/C, and CEL II were incubated at 42 ⁰ C for 20 minutes. The reaction was terminated by addition of 1/10 volume of 0.5 M EDTA. The DNA was analyzed by 1.5% agarose gel electrophoresis. Cutting of the mismatches in Control G/C produces two fragments 415- and 217-bp in length that are separated from the 632 -bp substrate during electrophoresis.

Culture supernatant from cells transfected with pcDNA3.1/CEL2 was fractionated on an HR 5/5 Mono-Q Column (Amersham Biosciences) . Elution fractions were tested for mismatch cutting activity. None was found.

Cells extracts from 293 cells transfected with pcDNA3.1/CEL2-Flag were fractionated by SDS PAGE, the proteins were transferred by Western blotting, and the blot was probed with antibody to Flag. No CEL II -Flag fusion protein responding to the antibody was observed. Example 3: Construction of pcDNA3.1/CEL2-His-V5 for Expression of CEL II in Cultured Human 293 Cells A pcDNA3.1/CEL2 -His-V5 expression construct was prepared. To introduce a better Kozak sequence

(underlined) upstream of the CEL II ORF to improve CEL II expression, the following sequence was used:

caccgccacc [ATG] GGTATGTTGACTTATACTGGAATTTATTTTC (SEQ ID NO:10). The C-terminus of the CEL II ORF was fused in frame with a His ₆ tag and V5 tag in the vector, so that the CEL II expression product could be concentrated and purified by Ni-affinity chromatography. The CEL II ORF sequences were PCR amplified with Optimase Polymerase using 5'-CACCGCCACCATGGGTATGTTGACTTATACTGGAATTTATTTTC-S' (SEQ ID NO : 10 ) and 5 ' -CACTATTTCAATATTGTTACTGCTTCTATGTC-3 ' (SEQ ID NO: 11) as the forward and the reverse primers. The PCR product was ligated to the vector and the construct was used to transform E. coli competent cells. Plasmid DNA was purified from a culture derived from a transformed colony with a QIAGEN DNA purification column. DNA sequencing of the CEL II gene insert showed the sequence to be 100% correct. The insert region sequence was:

5 ' -TTAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGTTAAGCTTGGTACCGAGC TCGGATCCAGTACCCTTCACCGCCACCATGGGTATGTTGACTTATACTGGAATTTATTT TCTGCTATTACTTCCAAGTGTTTTCTGTTGGGGAAAACAAGGACATTTTGCAATTTGTA AAATTGCCCAGGGGTTCCTTAGTAAAGATGCACTGACTGCAGTGAAAGCATTGCTCCCA GAATATGCAGATGGTGATCTAGCAGCTGTTTGCTCCTGGGCTGACGAGGTTCGATTTCA TATGCGTTGGAGTAGCCCATTACATTATGTGGACACGCCTGATTTCAGGTGTAACTATA AATACTGTAGAGATTGCCATGATTCTGTTGGACGGAAAGACCGGTGTGTTACTGGAGCA ATTCACAACTACACAGAGCAACTTCTATTGGGTGTTCATGACTTGAATTCAAAAATGAA TAACAACTTGACGGAGGCACTTATGTTCTTATCACATTTCGTTGGTGATGTCCATCAGC CTCTACATGTTGGCTTCCTTGGCGATGAAGGAGGAAACACAATCACCGTCCGCTGGTAT

CGGAGGAAAACCAATTTGCATCATGTATGGGACACAATGATGATTGAATCCTCCTTG AA GACATTCTACAATTCAGATCTTTCTAGCTTAATACAAGCTATTCAGAGCAATATTACAG GTGTCTGGCTTACCGACAGCTTATCTTGGAGCAATTGCACTGCTGATCATGTGGTTTGT CCAGACCCGTATGCTTCTGAAAGCATTGAGTTGGCCTGCAAGTTTGCCTACAGAAATGC CACACCTGGGACCACTTTAGGAGATGAGTACTTCCTCTCTCGGTTGCCTGTTGCGGAGA

AGAGGTTGGCTCAGGCTGGGGTCCGTTTGGCTGCTACTCTTAACCGAATCTTCACTT CA AACCCCAGCGATCTCACAAGATTGAATATGCATAATGGTGGACATAGAAGCAGTAACAA TATTGAAATAGTGAAGGGTCAAGACAATTCTGCAGATATCCAGCACAGTGGCGGCCGCT CGAGTCTAGAGGGCCCGCGGTTCGAAGGTAAGCCTATCCCTAACCCTCTCCTCGGTCTC GATTCTACGCGTACCGGTCATCATCACCATCACCATTGAGTTTAAACCCGCTGATCAGC CTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCT TGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAA-S' (SEQ ID NO: 12)

The protein sequence was :

MGMLTYTGIYFLLLLPSVFCWGKQGHFAICKIAQGFLSKDALTAVKALLPEYADGDL AA VCSWADEVRFHMRWSSPLHYVDTPDFRCNYKYCRDCHDSVGRKDRCVTGAIHNYTEQLL LGVHDLNSKMNNNLTEALMFLSHFVGDVHQPLHVGFLGDEGGNTITVRWYRRKTNLHHV WDTMMIESSLKTFYNSDLSSLIQAIQSNITGVWLTDSLSWSNCTADHWCPDPYASESI ELACKFAYRNATPGTTLGDEYFLSRLPVAEKRLAQAGVRLAATLNRIFTSNPSDLTRLN MHNGGHRSSNNIEIVkgqdnsadiqhsggrsslegprfeGKPIPNPLLGLDSTrtgHHH HHH (SEQ ID NO: 13)

In the above sequence, the signal peptide is underlined, the His ₆ tag is italicized, the V5 tag is bolded, and the sequence in lowercase letters is derived from the vector.

Example 4 : Transient Expression of CEL II in Cultured Human 293 Cells The plasmid DNA pcDNA3. l/CEL2-His-V5 was transiently transfected into 293T cells with Lipofectamine (Invitrogen) in 10-cm culture dishes. pUC19 plasmid DNA was used as a mock transfection control .

Aliquots (5, 25, 50 μL) of culture supernatants at 3- days post-transfection, as well as cell lysates, were assayed for mismatch cutting activity with Control G/C DNA.

No activity was observed.

The supernatant and the cell lysate were bound separately to Ni-NTA agarose beads (Qiagen) . After elution no mismatch cutting activity was found with Control G/C substrate in either purified culture supernatant or cell lysate .

Comparative SDS PAGE analysis of culture supernatants and cell extracts from 293 cells transfected with pcDNA3.1/CEL2-His-V5 DNA or with pUC19 DNA showed no new bands in the supernatant and cell lysate from cells transfected with pcDNA3. l/CEL2-His-V5 DNA relative to cells mock transfected.

Example 5: Positive Evidence for Successful Transfection of Cultured Human 293 Cells with Vectors Bearing the CEL II Gene

Because all results to this point were negative, including assays that should have detected the expression of any CEL II protein, active or inactive, experiments were performed to assess whether plasmid DNA was actually being transfected into 293 cells. pcDNA3.1-CEL2 , pcDNA3.1- CEL2.Flag and pcDNA3.1-CEL2/His-V5 plasmid DNAs were each co-transfected with pcDNA3. l-LacZ/His-V5 positive control plasmid. Cells were mock transfected with pcDNA3.1D/V5-His alone. If the co-transfected DNA, pcDNA3. l-LacZ/His-V5 , successfully entered cells, expression of β-galactosidase would ensue, indicating that co-transfected CEL II constructs entered the cells as well. Expression of β- galactosidase was assayed by fixing and staining some of the cells with X-GaI 72 hours after transfection. To stain the transfected cells with X-GaI, the cells were washed with Ix cold PBS, fixed with approximately 5 mL glutaraldehyde (1:100 dilution of 25% stock in PBS) for 5 minutes, and rinsed 3 times with PBS for 4 minutes. X-GaI staining solution (5 mL) was added to the cells and incubated at 37°C for 1-20 hours. The X-GaI staining solution was 50 μg MgCl ₂ , 41 mg potassium-ferricyanide (potassium hexacyanoferrate (III) ) , 53 mg potassium- ferrocyanide (potassium hexacyanoferrate (II) ), and 1 mg/mL X-GaI in 25 mL of PBS (pH 7.3). Stained cells were photographed under light microscopy. Mock-transfected cells showed no staining with X-GaI. Cells transfected with pcDNA3.1-LacZ/His-V5 alone showed intense staining with X- GaI, while cells co-transfected with pcDNA3. l-LacZ/His-V5 and any one of the constructs bearing the CEL II gene were

also stained by X-GaI, but to a diminished extent. These results clearly demonstrated that transfection of the 293 cells with pcDNA3.1 constructs was successful. Example 6 : Expression of Enzymatically Active Recombinant CEL II by Supplementing the Culture Medium with Zn ⁺⁺

ZnCl ₂ (10 μM) was added to the culture medium upon replacement of the transfection medium with culture medium 16 hours post -transfection. Seventy-two hours post transfection, the culture supernatants from transfected cell cultures were filtered with 0.2 micron filters and tested for the presence of CEL II enzymatic activity. In particular, supernatants were assayed for activity that cut heteroduplexes present in 200 ng of Control G/C heteroduplex DNA. Cell extracts were also prepared and assayed for activity. Supernatants and cell lysates (5 μL) from cells transfected with pcDNA3.1-LacZ/His-V5 , pcDNA3.1- CEL2, pcDNA3.1-CEL2. Flag, pcDNA3.1-CEL2/His-V5 or pcDNA3.1- His-V5 (mock transfected) were incubated with 5 μL (200 ng) of Control G/C and 1 μL SURVEYOR Nuclease Enhancer (Transgenomic, Inc.). SURVEYOR Nuclease S (Transgenomic, Inc.) was incubated separately as a positive control. The reactions were carried out at 42 ⁰ C for 20 minutes. CEL II mismatch cutting activity was observed, but only in the Zn ⁺⁺ -containing culture medium from pcDNA3.1-CEL2/His-V5 transfected cells.

Example 7: Purification of CEL II from Culture Supernatant from 293 Cells Transfected with pcDNA3.1-CEL2/His-V5

Three ml of culture supernatant from 293 cells transfected with pcDNA3.1-CEL2/His-V5 and grown in the presence of 10 μM ZnCl ₂ was harvested 72 hours post transfection and clarified by centrifugation (12,000 x g for 15 minutes) . The clarified supernatant was passed over

a 1-mL HiTRAP Ni -Chelating column (Amersham) equilibrated in Buffer A [20 mM KPO ₄ (pH 7.6) -0.5 M NaCl-10% glycerol] plus 5 mM imidazole. CEL II with a fused His ₆ tag binds to this column. Bound protein was eluted with a linear gradient in Buffer A plus 0.05 to 0.5 M imidazole. The nuclease activity in fractions was measured with two assays: solubilization of ssDNA and cutting of Control G/C mismatch DNA. No activity was found with either assay. Active CEL II could have been present, but not at measurable concentrations because: imidazole and high salt inhibit CEL II activity and the concentration of CEL II after gradient elution would have been low.

All the fractions from the chelating column were pooled and dialyzed against 50 mM Tris-HCl (pH 8.0) -5 mM α- methyl mannoside-0.01% Triton X-100-10 μM ZnCl ₂ . The dialyzed pool was bound to an HR 5/5 MonoQ column equilibrated in Buffer B [50 mM Tris-HCl (pH 8.0) -5 mM comethyl mannoside-0.01% Triton X-100] . The column was batch eluted with Buffer B plus 250 mM KCl. Two, 1 ml fractions were recovered and 1-μl aliquots of the fractions were assayed for mismatch cutting activity with Control G/C. A small amount of mismatch cutting activity was observed in each fraction. The 1 ml fractions were each concentrated to approximately 25 μl with a Microcon-10 (Amicon) . The concentrated samples were recovered and 100% glycerol was added to a final concentration of 50%. The Microcon membrane was rinsed with a small volume of Buffer A and the rinse was saved.

The concentrated fractions and rinses were assayed for mismatch cutting activity with a Control G/C heteroduplex substrate. Both fractions contained mismatch cutting activity, demonstrating the expression and secretion of

enzymatically active cloned CEL II with a carboxy-terminal His ₆ tag from human 293 cells grown in the presence of 10 μM ZnCl ₂ .

Example 8 : Generation of a Stable Cell Line Expressing Recombinant CEL II

Human 293F cells grown in a 10-cm diameter cell culture dish to 50% confluence were transfected with 12 μg of pcDNA3.1-CEL2/His-V5 DNA in 800 μL of OptiMEM (Invitrogen) mixed with 800 μL of OptiMEM containing 25 μL of Lipofectamine (Invitrogen). After 5 hours at 37 ⁰ C, 6.4 mL of OptiMEM was added and the dish was incubated overnight at 37°C in a 5% CO ₂ incubator. The cell ^' s were then treated with a 1:5 dilution of TrypLE Express

(Invitrogen) in 1 x PBS containing 0.53 mM EDTA (pH 8.0) at room temperature. The TrypLE Express was removed and the cells were suspended in 10 mL of DMEM medium supplemented with 10% horse serum, 2 mM glutamine, 50 units/mL penicillin, 50 μg/mL streptomycin, 10 μM ZnCl ₂ and 500 μg/mL Geneticin. Serial dilutions (1:3) of the suspension were made in supplemented DMEM medium and twelve, 200-μL aliquots of each dilution were dispensed into 12 wells of a 96-well cell culture plate. Wells in the dilution series containing less than 10 growing cells per well after the selection were expanded in a 6 -well plate in 4 mL of supplemented culture medium/well. The 6-well plate was incubated at 37°C for 7 days in a 5% CO ₂ incubator.

The culture supernatant (4 mL) from each well was filtered through a 0.2 micron filtration disc and mixed with 100 μL of Ni-NTA agarose (Qiagen) slurry for 30 minutes. The slurry was washed with 1 x CEL II Reaction Buffer [20 mM Tris-HCl (pH 7.4), 25 mM KCl, 10 mM MgCl ₂ , and 10 μM ZnCl ₂ ] and eluted in 50 μL of 300 mM imidazole (pH

8.0) in 1 x CEL II Reaction Buffer. The eluant was filtered through a G-25 Microspin column (Amersham Biosciences) . Filtered eluant (10 μL) was tested for mismatch cutting activity with Control G/C DNA as described in Example 2 (Figure 1) . The B-6 clone was selected for cell growth on a larger scale.

For expression of recombinant CEL II on a liter scale, the B-6 clone was cultured in twenty, 15-cm diameter culture dishes in 50 mL/dish DMEM medium supplemented with 0.2% horse serum, 2 mM glutamine, 10 μM ZnCl ₂ and antibiotics. After approximately 10 days in culture, the supernatant was harvested by spinning down the cells at 1,000 x g and filtering the supernatant with a 0.2 micron filtration unit. The supernatant was tested for CEL II activity by mixing 5 mL with 100 μL of Ni-NTA agarose beads, eluting with 50 μL of 300 mM imidazole in IX CEL II Reaction Buffer, and testing 10 μL of the eluant for mismatch cutting activity with Control -G/C heteroduplex. The B-6 expanded clonal cell line clearly expressed enzymatically active CEL II (Figure 2) . The cells recovered by centrifugation were resuspended in 1 liter of medium for continued propagation in dishes to produce more CEL II. The expression level was undiminished after 3 passages.

The B-6 clonal cells were also adapted to spinner suspension culture. A frozen vial of B-6 cells was thawed into 50 mL DMEM supplemented with 10% serum, 50 unit/mL penicillin, 50 μg/mL streptomycin, and 2 mM glutamine in a 15-cm dish. When the cells became subconfluent , the cell monolayer was rinsed with 10 mL of a 1:5 dilution of TrypLE Express in 0.53 mM EDTA in 1 x PBS. The cells were resuspended in 1 liter of 293 SFMII IX liquid (Invitrogen) , supplemented with 4 mM glutamine, penicillin/streptomycin

and 20 μM ZnCl ₂ in a 2-liter spinner. After one week in culture in a humidified 8% CO ₂ incubator at 37°C, the cells were recovered by centrifugation at 1,000 x g. An aliquot

(5 mL) of culture supernatant was taken, filtered, and mixed with 100 μL of Ni-NTA agarose beads. The beads were eluted with 50 μL of 300 mM imidazole in IX CEL II Reaction

Buffer. An aliquot of the eluant (5 μL) was assayed for mismatch cutting activity with Control G/C and found to contain activity (Figure 3) . The cells recovered from centrifugation of the spinner culture were resuspended in 1 liter of DMEM supplemented with 0.2% horse serum, 50 unit/mL penicillin, 50 μg/mL streptomycin, 4 mM glutamine, and 20 μM ZnCl ₂ . The cells were grown in spinner culture at 37°C in a humidified 8% CO ₂ incubator. The culture supernatant was again assayed for CEL II mismatch cutting activity as already described. Activity was present at a level comparable to that in the original spinner culture supernatant (Figure 4) . Example 9: Purification and Characterization of CEL II from Culture Supernatant from a Permanent 293 Cell Line Expressing Recombinant CEL II

A liter of culture supernatant from the B-6 clonal 293 cell line grown in 15-cm cell culture dishes was clarified by centrifugation (1,000 x g for 15 min) and filtered (0.45 micron filter) . The filtered supernatant was adjusted in pH by the addition of 1 M Tris-HCl (pH 8.0) to a final concentration of 100 mM. CEL II was precipitated by the addition of 619 gms of solid ammonium sulfate and centrifugation at 4 ⁰ C at 14,000 x g for 60 min. The precipitate was dissolved in 40 mL of Buffer C [50 mM Tris- HCl (pH 8.0) - 0.01% Triton X-100] + 0.1 M NaCl and clarified by centrifugation (12,000 x g; 20 min) . The

clarified supernatant was passed over a 5-mL Ni-Chelating column (Amersham Biosciences) equilibrated in Buffer C + 0.1 M NaCl. The recombinant CEL II was eluted with Buffer C + 0.1 M NaCl + 0.3 M imidazole. Fractions containing protein were pooled. Approximately 120,000 units of CEL II activity was recovered as determined by the single-stranded DNA solubilization assay (see Example 2 for assay) . The pooled fractions from the Ni-Chelating column were dialyzed against Buffer D [20 mM NaOAc (pH 5.0) -0.1 mM ZnSO ₄ -0.01% Triton X-100] overnight and clarified by centrifugation at 12,000 x g for 20 min. The dialyzed pool was loaded unto a 5-mL Hi-TRAP Heparin column (Amersham Biosciences) equilibrated in Buffer D + 50 mM α-methyl mannoside. The column was washed with Buffer D + 50 mM α-methyl mannoside and eluted with a linear gradient of 0.15 M to 0.8 M KCl in Buffer D + 50 mM α-methyl mannoside. Fractions were assayed for single-stranded DNase activity and peak activity fractions were pooled and the pH of the pool was adjusted to 8.0 by addition of 1 M Tris-HCl (pH 8.0). The pooled peak fractions were dialyzed against 500 mL of Buffer E [20 mM KPO ₄ (pH 7.O)-IO μM ZnCl ₂ -0.01% Triton X-100] overnight. The dialyzed pool was loaded unto a 1-mL Hi-TRAP Heparin column (Amersham Biosciences) equilibrated in Buffer E. CEL II was eluted with Buffer E + 0.3 M KCl. Fractions were assayed for single-stranded DNase activity and peak activity fractions were pooled and dialyzed against Storage Buffer [50 mM Tris-HCl (pH 7.5) -100 mM KCl-10 μM ZnCl ₂ -O.01% (v/v) Triton X-100-50% (v/v) glycerol] . Approximately 30,000 units of recombinant CEL II were recovered. Recombinant CEL II had enzymatic properties similar to native CEL II. The pH optimum of recombinant CEL II was ~9.0 as determined by mismatch cutting efficiency of Control G/C heteroduplexes, similar to native CEL II.

Recombinant CEL II was like native CEL II in ability to cut A/G and C/T mismatches embedded in sequence contexts which make the mismatches difficult to cut.