Backaround Information The bacteriophage been studied extensively, and, as a result, much is known about its structure and function.

The Xphage andXcosmid have become standard viral vectors for use in cloning and characterization of genes. One enzyme essential for several aspects of packaging of viral DNA (with or without foreign DNA) into empty proheads is the "terminase"enzyme. In the lab, theXterminase enzyme is widely used in preparation of genomic libraries inXphage and cosmid vectors and analyis of genomic libraries that are in cosmid vectors.

Prior to the present invention, however, Xterminase was difficult to purify. It was historically purified using three columns, the first being SP-sepharose fast flow chromatography column, the second being a Q-sepharose fast flow chromatography column, and the third being a blue- sepharose fast flow chromatography column. The yield was

around 1-2 mg from 8000 mg of starting protein. The difficulty of purification was reflected in the cost, and, in the case of cosmid clone analysis, often proved prohibitively expensive.

One method for purification of proteins with known DNA sequences has been called"histidine tagging."In this method, several histidine codons are inserted at a terminus of the gene encoding the protein of interest, the gene is subsequently expressed in host cells, and the lysate of the host cells is then loaded onto a column with available nickel ions. The histidines selectively bind to the nickel ions and the protein is thereby purified.

Histidine tagging of proteins has been done in the past, with reported success. According to Current Protocols in Molecular Biology, Supp 36.10.11.22 (1997),"Histidine tail fusion proteins often retain their normal biologic functions..."and cites histidine-tagged dihydrofoloate reductase, adenylcyclase and the TATA box binding protein as examples. Hochuli, 12 Gen. Eng. Princ. & Prac. 87 (1990); Taussig et al., 268 J. Biol. Chem. 9 (1993); and Parvin et al., 68 Cell 1135 (1992). In this statement, the authors use circular reasoning: successfully His-tagged proteins are used as evidence of success retaining function in His- tagging proteins. The flawed reasoning is no fault of the authors, since only positive results are accepted for publication in scientific journals. In other words,

negative results were not publishable, nor published. In every day practice in the laboratory, the experience of many researchers has been that purification of proteins via His tagging is unpredictable.

One piece of evidence of the unpredictability of His tagging as a purification method is the existence of ocher purification schemes. For example, many commercial kits are on the market for purifying proteins with known sequences, for example: maltose binding protein systems, such as the Protein Fusion and Purification System of New England Biolabs, peptide tag systems involving antibody recognition, like the FLAG system of IBI/Kodak, self-cleavable affinity tag systems such as the Impact I system of New England Biolabs, and the glutathione-S-transferase systems such as that of Pharmacia Biotech.

In fact, Current Protocols has a"troubleshooting" section, wherein the authors describe the most common problem as low yield. Supplement 36 10.11.22, right column (1997). One of the reasons given is that"the histidine tail is buried in the protein and is inaccessible to the resin," (10.11.23, left column) and suggests purifying "under denaturing conditions"or placement"of the histidine tail at the opposite terminus." Current Protocols elucidates two problems associated with histidine tagging:

1.) inavailability of the histidine tag to binding by the nickel column; and 2.) inactivity/loss of function of a protein with extra histidines on the terminal end.

Citation of the above documents is not intended as an admission that any of the foregoing is pertinent prior art. For example, in some instances above, the publication was less than one year before the filing date of this patent application. All statements as to the date or representation as to the contents of these documents are based on subjective characterization of information available to the applicant at the time of filing, and do not constitute an admission as to the accuracy of the dates or contents of these documents.

SUMMARY OF THE INVENTION It is therefore an object of the present invention to provide compounds which can be expressed into easily purifiable enzymes with terminase activity.

It is a further object to provide modified terminase enzymes which are easily purified and retain terminase activity.

It is yet another object to provide methods for purifying modified terminase enzymes.

It is yet another object to provide methods for

packaging DNA using a modified terminase enzymes.

It is yet another object to provide methods for preparing and analyzing vectors using modified terminase enzymes.

It is yet another object to provide methods for preparing X cosmid libraries using modified terminase enzymes.

For the purposed of this application, the following words shall have the following meanings: "Histidine analog"includes any compound with similiar ionic and structural features to histidine, including histidine itself.

"Modified and active lambda terminase enzymes used interchangeably with"His tagged enzyme"or variants on those two terms. The meaning is intended to be that of a lambda terminase enzyme which has a series of histidines at the carboxy end of the gpAgene, as described herein.

"Strong concentration of a histidine analogue"a concentration of a histidine analogue which would result in disassociation of a His-tagged terminase from a metal cation when put in contact with the His-tagged terminase/metal cation complex.

"Weak concentration of a histidine analogue concentration of a histidine analogue which would not result in disassociation of a His-tagged terminase from a metal

cation cation when put in contact with the His-tagged terminase/metal cation complex.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides, inter alia, modified and active lambda terminase enzymes, wherein the modification is a histidine tag in or at the carboxy end of the gene product A subunit (gpA), the product of the A gene, as described herein. Specifically, the histidine tag can be at the end of the gpA sequence, or in place of the last 1, <BR> <BR> <BR> <BR> <BR> <BR> <BR> 2,3 or 4 codons. The stop codon should remain unaffected by the alteration, and is not considered part of the carboxy end of the gpA sequence. Preferrably, the histidine codons are at the end of the gpA sequence, or in place of the last 1,2 or 3 codons. The most preferred embodiment is one wherein the histidine tag is at the end of the gpA sequence.

The number of histidine residues is important only to meet the purpose of sufficient binding to the metal cation.

Six histidine residues is acceptable for the present invention, although those in the art recognize that fewer or especially more histidine residues would work in this method. Therefore, 1 to 10 residues may be used in the present invention, but 4 to 8 are preferred. The most preferred embodiment is one in which six residues are used.

Also, the tag need not contain exclusively histidine residues, so long as the binding function is retained. For

instance, alanine residue (s) can be included, if desired.

In particular, there are provided modified and active lambda terminase having the following sequences: SEQ ID Sequence NO 1 1 MNISNSQVNR LRHFVRAGLR SLFRPEPQTA VEWADANYYL PKESAYQEGR 51 WETLPFQRAI MNAMGSDYIR EVNVVKSARV GYSKMLLGVY AYFIEHKQRN 101 TLIWLPTDGD AENFMKTHVE PTIRDIPSLL ALAPWYGKKH RDNTLTMKRF 151 TNGRGFWCLG GKAAKNYREK SVDVAGYDEL AAFDDDIEQE GSPTFLGDKR 201 IEGSVWPKSI RGSTPKVRGT CQIERAASES PHFMRFHVAC PHCGEEQYLK 251 FGDKETPFGL KWTPDDPSSV FYLCEHNACV IRQQELDFTD ARYICEKTGI 301 WTRDGILWFS SSGEEIEPPD SVTFHIWTAY SPFTTWVQIV KDWMKTKGDT 351 GKRKTFVNTT LGETWEAKIG ERPDAEVMAE RKEHYSAPVP DRVAYLTAGI 401 DSQLDRYEMR VWGWGPGEES WLIDRQIIMG RHDDEQTLLR VDEAINKTYT 451 RRNGAEMSIS RICWDTGGID PTIVYERSKK HGLFRVIPIK GASVYGKPVA 501 SMPRKRNKNG VYLTEIGTDT AKEQIYNRFT LTPEGDEPLP GAVHFPNNPD 551 IFDLTEAQQL TAEEQVEKWV DGRKKILWDS KKRRNEALDC FVYALAALRI 601 SISRWQLDLS ALLASLQEED GAATNKKTLA DYARALSGED EHHHHHH 2 1 MNISNSQVNR LRHFVRAGLR SLFRPEPQTA VEWADANYYL PKESAYQEGR 51 WETLPFQRAI MNAMGSDYIR EVNVVKSARV GYSKMLLGVY AYFIEHKQRN 101 TLIWLPTDGD AENFMKTHVE PTIRDIPSLL ALAPWYGKKH RDNTLTMKRF 151 TNGRGFWCLG GKAAKNYREK SVDVAGYDEL AAFDDDIEQE GSPTFLGDKR 201 IEGSVWPKSI RGSTPKVRGT CQIERAASES PHFMRFHVAC PHCGEEQYLK 251 FGDKETPFGL KWTPDDPSSV FYLCEHNACV IRQQELDFTD ARYICEKTGI 301 WTRDGILWFS SSGEEIEPPD SVTFHIWTAY SPFTTWVQIV KDWMKTKGDT 351 GKRKTFVNTT LGETWEAKIG ERPDAEVMAE RKEHYSAPVP DRVAYLTAGI

401 DSQLDRYEMR VWGWGPGEES WLIDRQIIMG RHDDEQTLLR VDEAINKTYT 451 RRNGAEMSIS RICWDTGGID PTIVYERSKK HGLFRVIPIK GASVYGKPVA 501 SMPRKRNKNG VYLTEIGTDT AKEQIYNRFT LTPEGDEPLP GAVHFPNNPD 551 IFDLTEAQQL TAEEQVEKWV DGRKKILWDS KKRRNEALDC FVYALAALRI 601 SISRWQLDLS ALLASLQEED GAATNKKTLA DYARALSGED HHHHHH 3 1 MNISNSQVNR LRHFVRAGLR SLFRPEPQTA VEWADANYYL PKESAYQEGR 51 WETLPFQRAI MNAMGSDYIR EVNVVKSARV GYSKMLLGVY AYFIEHKQRN 101 TLIWLPTDGD AENFMKTHVE PTIRDIPSLL ALAPWYGKKH RDNTLTMKRF 151 TNGRGFWCLG GKAAKNYREK SVDVAGYDEL AAFDDDIEQE GSPTFLGDKR 201 IEGSVWPKSI RGSTPKVRGT CQIERAASES PHFMRFHVAC PHCGEEQYLK 251 FGDKETPFGL KWTPDDPSSV FYLCEHNACV IRQQELDFTD ARYICEKTGI 301 WTRDGILWFS SSGEEIEPPD SVTFHIWTAY SPFTTWVQIV KDWMKTKGDT 351 GKRKTFVNTT LGETWEAKIG ERPDAEVMAE RKEHYSAPVP DRVAYLTAGI 401 DSQLDRYEMR V ; iGWGPGEES WLIDRQIIMG RHDDEQTLLR VDEAINKTYT 451 RRNGAEMSIS RICWDTGGID PTIVYERSKK HGLFRVIPIK GASVYGKPVA 501 SMPRKRNKNG VYLTEIGTDT AKEQIYNRFT LTPEGDEPLP GAVHFPNNPD 551 IFDLTEAQQL TAEEQVEKWV DGRKKILWDS KKRRNEALDC FVYALAALRI 601 SISRWQLDLS ALLASLQEED GAATNKKTLA DYARALSGEH HHHHH 4 1 MNISNSQVNR LRHFVRAGLR SLFRPEPQTA VEWADANYYL PKESAYQEGR 51 WETLPFQRAI MNAMGSDYIR EVNVVKSARV GYSKMLLGVY AYFIEHKQRN 101 TLIWLPTDGD AENFMKTHVE PTIRDIPSLL ALAPWYGKKH RDNTLTMKRF 151 TNGRGFWCLG GKAAKNYREK SVDVAGYDEL AAFDDDIEQE GSPTFLGDKR 201 IEGSVWPKSI RGSTPKVRGT CQIERAASES PHFMRFHVAC PHCGEEQYLK 251 FGDKETPFGL KWTPDDPSSV FYLCEHNACV IRQQELDFTD ARYICEKTGI

301 WTRDGILWFS SSGEEIEPPD SVTFHIWTAY SPFTTWVQIV KDWMKTKGDT 351 GKRKTFVNTT LGETWEAKIG ERPDAEVMAE RKEHYSAPVP DRVAYLTAGI 401 DSQLDRYEMR VWGWGPGEES WLIDRQIIMG RHDDEQTLLR VDEAINKTYT 451 RRNGAEMSIS RICWDTGGID PTIVYERSKK HGLFRVIPIK GASVYGKPVA 501 SMPRKRNKNG VYLTEIGTDT AKEQIYNRFT LTPEGDEPLP GAVHFPNNPD 551 IFDLTEAQQL TAEEQVEKWV DGRKKILWDS KKRRNEALDC FVYALAALRI 601 SISRWQLDLS ALLASLQEED GAATNKKTLA DYARALSGHH HHHH The present invention therefore also provides DMA compounds which encode the modified and active lambda terminase enzymes. Specifically disclosed are DNA compounds which encode SEQ ID Nos 1-4. Preferred DNA compounds of the present invention is SEQ ID NO 5: ATGAATATAT CGAACAGTCA GGTTAACAGG CTGCGGCATT TTGTCCGCGC CGGGCTTCGC TCACTGTTCA GGCCGGAGCC ACAGACCGCC GTTGAATGGG CGGATGCTAA TTACTATCTC CCGAAAGAAT CCGCATACCA GGAAGGGCGC TGGGAAACAC TGCCCTTTCA GCGGGCCATC ATGAATGCGA TGGGCAGCGA CTACATCCGT GAGGTGAATG TGGTGAAGTC TGCCCGTGTC GGTTATTCCA AAATGCTGCT GGGTGTTTAT GCCTACTTTA TAGAGCATAA GCAGCGCAAC ACCCTTATCT GGTTGCCGAC GGATGGTGAT GCCGAGAACT TTATGAAAAC CCACGTTGAG CCGACTATTC GTGATATTCC GTCGCTGCTG GCGCTGGCCC CGTGGTATGG CAAAAAGCAC CGGGATAACA CGCTCACCAT GAAGCGTTTC ACTAATGGGC GTGGCTTCTG GTGCCTGGGC GGTAAAGCGG CAAAAAACTA CCGTGAAAAG TCGGTGGATG TGGCGGGTTA TGATGAACTT GCTGCTTTTG ATGATGATAT TGAACAGGAA GGCTCTCCGA CGTTCCTGGG TGACAAGCGT ATTGAAGGCT CGGTCTGGCC AAAGTCCATC CGTGGCTCCA

CGCCAAAAGT GAGAGGCACC TGTCAGATTG AGCGTGCAGCCAGTGAATCC CCGCATTTTA TGCGTTTTCA TGTTGCCTGC CCGCATTGCG GGGAGGAGCA GTATCTTAAA TTTGGCGACA AAGAGACGCC GTTTGGCCTC AAATGGACGC CGGATGACCC CTCCAGCGTG TTTTATCTCT GCGAGCATAA TGCCTGCGTC ATCCGCCAGC AGGAGCTGGA CTTTACTGAT GCCCGTTATA TCTGCGAAAA GACCGGGATC TGGACCCGTG ATGGCATTCT CTGGTTTTCG TCATCCGGTG AAGAGATTGA GCCACCTGAC AGTGTGACCT TTCACATCTG GACAGCGTAC AGCCCGTTCA CCACCTGGGT GCAGATTGTC AAAGACTGGA TGAAAACGAA AGGGGATACG GGAAAACGTA AAACCTTCGT AAACACCACG CTCGGTGAGA CGTGGGAGGC GAAAATTGGC GAACGTCCGG ATGCTGAAGT GATGGCAGAG CGGAAAGAGC ATTATTCAGC GCCCGTTCCT GACCGTGTGG CTTACCTGAC CGCCGGTATC GACTCCCAGC TGGACCGCTA CGAAATGCGC GTATGGGGAT GGGGGCCGGG TGAGGAAAGC TGGCTGATTG ACCGGCAGAT TATTATGGGC CGCCACGACG ATGAACAGAC GCTGCTGCGT GTGGATGAGG CCATCAATAA AACCTATACC CGCCGGAATG GTGCAGAAAT GTCGATATCC CGTATCTGCT GGGATACI'GG CGGGATTGAC CCGACCATTG TGTATGAACG CTCGAAAAAA CATGGGCTGT TCCGGGTGAT CCCCATTAAA GGGGCATCCG TCTACGGAAA GCCGGTGGCC AGCATGCCAC GTAAGCGAAA CAAAAACGGG GTTTACCTTA CCGAAATCGG TACGGATACC GCGAAAGAGC AGATTTATAA CCGCTTCACA CTGACGCCGG AAGGGGATGA ACCGCTTCCC GGTGCCGTTC ACTTCCCGAA TAACCCGGAT ATTTTTGATC TGACCGAAGC GCAGCAGCTG ACTGCTGAAG AGCAGGTCGA AAAATGGGTG GATGGCAGGA AAAAAATACT GTGGGACAGC AAAAAGCGAC GCAATGAGGC ACTCGACTGC TTCGTTTATG CGCTGGCGGC GCTGCGCATC AGTATTTCCC GCTGGCAGCT GGATCTCAGT GCGCTGCTGG CGAGCCTGCA GGAAGAGGAT GGTGCAGCAA CCAACAAGAA AACACTGGCA GATTACGCCC GTGCCTTATC CGGAGAGGAT GAACACCATC ACCATCACCAT

The amino acid and DNA compounds of the present invention may be obtained by conventional molecular biology methods. In particular, the DNA compounds may be made by partial digestion of the lambda plasmid so as to produce a restriction site in the desired region of the gpA sequence, (or sites may be created-creation of an AflII site is particularly useful) and subsequent ligation of an oligo with correspondingly-digested restriction sites. Some sites which are useful in this region are: XbaI and BspEI.

Alternatively, the sequences may be synthetically produced using automated sequencers, such as that made by ABI PRISM (Applied Biosystems, Inc.). Other methods are known in the art.

The histidine residues may be encoded by either sequence for histidine: CAC or CAT. Preferably, the sequences are alternated or at least mixed, so as to avoid recombination events. Skilled artisans are aware of the strategies in this regard, and also that, for short sequences, recombination is not as likely as for longer sequences. Thus, for shorter sequences, a few CAC sequences, for example, without intervening CAT sequences, would be acceptable, whereas for longer sequences, His- encoding sequences are optimally interspersed with each other.

The amino acid compounds may be obtained by expression of any of the DNA compounds above using conventional expression protocols such as that described in Sambrook et al, Molecular Cloning, Cold Spring Harbor Laboratory Press, (2nd Edition, 1989).

For example, the present compounds may be expressed, according to Chow et al., 60 Gene 277 (1987) as follows: The commercially available vector pETlld (Novagen, Inc.) can be used as a starting vector into which three constructions can be inserted, namely: (1) the Nul gene, (2) the amino-terminus of the A gene, and (3) the C-terminus of the A gene.

As discussed in Chow, the Nul and A genes of k have very poor ribosome binding sites (Shine-Dalgarno sequences), and so they must be replaced in this type of procedure. To replace the ribosome binding sites, the Nul and A genes, which normally overlap, can be separated. pETlld contains a strong ribosome binding site, followed by an Ncol restriction enzyme site that can be used to insert a gene.

The NcoI site is then used to insert the Nul gene. Because terminase is toxic, expression of terminase must be very low until induction. pETlld is designed to give very low expression under non-induced conditions.

The Nul gene can then be generated by the polymerase chain reaction (PCR) using commercialXDNA as template.

Primers such as: 5'-GTGGAATGAA CCATGGAAGT CACA-3' (SEQ ID NO 6) and 3'-ACGCCGTA AAACAGGTAC CGCCCGAAGC GAGTGA-5' (SEQ ID NO 7) which are complementary to the 5'and 3'termini of the Nul gene can be used. Note that each primer has a 5' tail containing an NcoI site. Following the PCR reaction, the product DNA segment and the vector pETlld can be digested with NcoI. The NcoI-digested DNAs can then be ligated together using T4 ligase. The ligation mixture may be used to transform competent E. coli cells to ampicillin resistance (ApR). Commercially available competent cells can be used, such as XL1-Blue cells (Stratagene, Inc.).

Plasmids can then be isolated from ApR transformants, and the plasmid DNA analyzed by restriction enzyme and sequence analysis to identify a derivative of pETlld containing the Nul gene in proper orientation: for the purposes of this application, this plasmid will be called pONE.

The 5'two-thirds of gene A can next be cloned into the commercial vector YEp24. The plasmid can replicate in E. coli due to the presence of the pBR322 origin of replication. CommercialX DNA can then be digested with HpaI and Sse83871 DNA. The fragment extending from the HpaI site at 734 to the Sse83871 site at 2561 can be isolated and ligated with YEp24 (also digested with HpaI and Sse8387I).

[Restriction enzyme site positions are given as the first base pair to the 5'side of the cut site on the top stand of

1 DNA.] YEp24 can be obtained from New England Biolabs, Inc. The ligated DNAs can then be used to transform competent E. coli cells to ampicillin resistance (ApR).

Plasmids can be isolated from ApR transformants, and the plasmid DNA analyzed by restriction enzyme and sequence analysis to identify a plasmid containing the gene A segment. The resulting plasmid can be digested with HpaI, a synthetic DNA segment produced by annealing of oligonucleotides: 5'-GACATAT GGAGGAGTCC ATGAATATAT CGAACAGTCA GGTT-3' (SEQ ID NO 8) and 3'-CTGTATA CCTCCTCAGG TACTTATATA GCTTGTCAGT CCAA-5' (SEQ ID NO 9) can be added, and the DNAs ligated. The ligated DNAs can then be used to transform competent E. coli cells to ampicillin resistance (ApR). Plasmids can then be isolated from ApR transformants, and the plasmid DNA analyzed by restriction enzyme and sequence analysis to identify a plasmid containing the synthetic DNA segment. The synthetic DNA segment completes the amino terminus of the A gene, adds a strong ribosome binding segment (that of the X FI gene), adds an NdeI site, and completes the HpaI site of Yep24: for the purpose of this application, this plasmid will be called pTWO.

The Sse83871 segment extending fromXbp 2561 to the site at 2824 can then be inserted into the Sse83871 site of YEp24. The ligated DNAs can be used to transform competent

E. coli cells to ampicillin resistance (ApR). Plasmids can be isolated from ApR transformants, and the plasmid DNA analyzed by restriction enzyme and sequence analysis to identify a plasmid containing the proper gene A segment.

The resulting plasmid can then be digested with BspEI to open the site at bp 2619, the synthetic DNA segment produced by annealing oligonucleotides 5'-CCGGAGAGG ATGAACACCA TCACCATCAC CATTGACGCG ACAGGATCCA CTT-3' (SEQ ID NO 10) and 3'- TCTCC TACTTGTGGT AGTGGTAGTG GTAACTGCGC TGTCCTAGGT GAAGGCC- 5' (SEQ ID NO 11) added, and the DNAs ligated together. The ligated DNAs can then be used to transform competent E. coli cells to ApR. Plasmids can be isolated from ApR transformants, and the plasmid DNA analyzed by restriction enzyme and sequence analysis to identify a plasmid containing the synthetic insert in proper orientation. The insert provides the terminus of the A gene and adds six histidine codons followed by a translation stop codon and a BamHI site: for the purpose of this application, this plasmid will be called pTHREE.

To assemble the expression vector, the Sse83871 segment extending from k bp 2561 to the site at 2824 in plasmid pTHREE can be isolated and inserted into Sse83871-digested pTWO. The ligated DNAs can be used to transform competent E. coli cells to ApR. Plasmids can be isolated from ApR transformants, and the plasmid DNA analyzed by restriction

enzyme and sequence analysis to identify a plasmid containing the Sse8387I segment from pTHREE in the proper orientation: for the purpose of this application, this plasmid is called pFOUR. pFOUR can then be digested with NdeI and BamHI and the modified A gene mixed with pONE, digested with NdeI and BamHI, and the DNAs ligated. The ligated DNAs can be used to transform competent E. coli cells to ApR. Plasmids can be isolated from ApR transformants, and the plasmid DNAs can then be analyzed by restriction enzyme and sequence analysis to identify a plasmid containing the segment from pFOUR: for the purposes of this application, this plasmid is called pFIVE and it is the complete expression vector for His- tagged terminase. pFIVE can be placed into BL21 (DE3) or BL21 (DE3) pLys cells (Stratagene, Inc.). Expression can be obtained using Stratagene protocols supplied with the BL21 (DE3) cells.

Furthermore, the present invention provides methods to purify His-tagged terminase enzymes. One such method comprises the steps of 1) combining lysed cell supernatant containing a His-tagged terminase enzyme with metal cation beads; 2) washing the combination with a weak concentration of a histidine analogue so as to leave His-tagged terminase attached to the metal cation beads; and 3) washing the His- tagged terminase and beads with a strong concentration of histidine analogue so as to remove the His-tagged terminase

from the metal cation beads.

Another method for purifying the compounds of the present invention comprises the steps of 1) lysing cells in which His-tagged terminase has been expressed ; 2) loading the lysate onto an immobilized metal cation column; 3) eluting the bound compound from the immobilized metal cation using a histidine analog.

Preferred metal cations for use in these methods are nickel and zinc. A most preferred purification method is one wherein the metal cation is nickel and the histidine analog is imidizole. However, imidizole may be used in any of the present methods as the histidine analog. Of course, histidine may be used as the histidine analogue as well.

Metal cation purification columns may be made by preloading a column with nickel beads. In particular, the FPLC HR10x10 (Pharmacia) column is useful in the present invention. Imidizole and histidine, for example, may be obtained from Sigma Chemical Company. Nickel and zinc beads may be obtained from Qiagen.

When determining the strength of the histidine analogue solution, the present invention is particularly effective when the following concentrations are used for the weak concentration wash: 5 mM, and the following concentrations are used for the strong concentrations wash: 150 mM.

However, the weak concentration wash can be from 2 mM to 20

mM and the strong concentration wash can be from 80 mM to 300 mM and still result in purified protein.

Also provided by the present invention is a method to package X phage into proheads, comprising mixing DNA, proheads and ATP with His-tagged terminase enzyme. Skilled artisans are aware of metal cation purification procedures.

Current Protocols, supplement 36 Unit 10.11B (1997) describes this procedure in detail.

Lambda proheads may be obtained commercially from Stratagene. ATP can be obtained from Boehringer Mannheim, and DNA may be obtained from various commercial sources (ie.

New England Biolabs). Skilled artisans are aware of lambda packaging procedures. An overview of these techniques can be found in Sambrook et al., at 3.27-3.43.

In another embodiment of the present invention, there is provided a method to cut DNA comprising mixing cos-site containing DNA and His-tagged terminase enzyme, according to skill of the art.

Deposit information The invention as claimed is enabled in the absences of the following information. However, since the following information is available, Applicants disclose it for the general benefit of the public after the expiration of any patent to issue on this application. On October 31,1997

Applicants deposited with the American Type Culture Collection, Rockville, Md, USA (ATCC) the materials described below: Plasmid pQH70, ATCC accession number 98562 This deposit was made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure and the regulations thereunder (Budapest Treaty).

This assures maintenance of a viable culture for 30 years from date of deposit. The organisms will be made available by ATCC under the terms of the Budapest Treaty, and subject to an agreement between Applicants and ATCC which assures unrestricted availability upon issuance of the pertinent U. S. patent. Availability of the deposited materials is not to be construed as a license to practice the invention in contravention of the rights granted under the authority of any government in accordance with its intellectual pronerty laws.

Examples Example 1. Construction of the altered gene.

A plasmid with an AflII site at 2615 and the A gene ("pQH70", ATCC accession number 98562) was cut with BspE1 (New England Biolabs,"NEB") and AflII (NEB), the AflII sticky ends trimmed with Mung Bean nuclease (NEB) so as to provide for expression of the His tag in the correct reading frame. The synthetic DNA segment containing the codons for

Leu-Ser-Gly-Glu-Asp-Glu-His-His-His-His-His-His (SEQ ID NO 12) (Integrated DNA Technologies, Inc.) was ligated with the larger fragment of pQH70 to form a plasmid which was then called pQH74. This addition completed the A gene and added six histidine codons.

Another vector pQH83 was then prepared to incorporate lambda sequences (lambda +2637 to +3329) flanking the A gene so that the A gene could then be subcloned into an expression vector. The sequence numbering is according to Daniels, Lambda II 519 (Cold Spring Harbor Lab Press 1983).

The expression vector pCM101 (from Helios Murialdo at the University of Toronto) and pQH83 were cut by Sse83871 (Panvera) and the appropriate fragments were ligated using DNA ligase.

Example 2. Expression of the altered gene.

100 ml broth was innoculated with E. coli cells, allowed to grow overnight at 37 degrees Celcius, with shaking. The culture was then transferred to larger flask, grown to log phase at 37 degrees Celcius. The culture was then subjected to a temperature of 45 degrees Celcius for 20 minutes and 40 degrees Celcius for one hour. The thus heat- induced cells were then pelleted and saved at-70 degrees Celsius until purification.

Example 3. FPLC Purification of the His-tagged protein.

The following protocol was used to purify greater than 8 mg His-tagged protein using Fine Precision Liquid Chromatography, and resulted in 4 to 10 times yields known prior to the present invention: Pack the HR 10x10 column (Pharmacia) with 7ml Ni-NTA superflow beads (Qiagen).

Equilibrate the column with 200ml of buffer F.

Load the column with terminase supernatant. The loading speed is 0.5m./min. [8Li (or more) of cell culture is spun down and suspended in 180ml (scale up corespondingly) of buffer with protease inhibitors PMSF and apoprotinin. After sonication, the terminase supernatant is obtained by centrifugation to remove the cell debris. Total protein was 1200 mg.) Wash the column with 100 ml buffer F + 5mM imidazole at 2ml/min.

Elute the column at lml/min. A linear gradient is applied first from 0 to 150 mM imidizole in buffer F for 10 min., and then the buffer F with 150mM imidizole is continuously applied for 60 min.

Collect the fractions and run SDS-PAGE to detect the purity of His6-terminase.

Dialysis against with buffer A+10% glycerol overnight, and then change with buffer A + 50% glycerol.

Store in-70 degree C.

Buffer A 20mM Tris*Hcl (pH9.0) lrE EDTA 10% glycerol (or 50% glycerol for storage of terminase) 7mM S-mercaptoethanol Buffer F 50mM Tris-HC1 (pH7.9) 0.5MI EDTA 500mM NaCl 10% glycerol up to 100ml with ds H20 Example 4. Nickel Bead Purification of the His-Tagged Terminase The following protocol was used to purify His-tagged terminase, and resulted in 20% purification of the terminase in the supernatant: Pellet 20ml induced culture by centrifuge.

Suspend in 1ml buffer F+ 500 mM NaCl.

(*The supernatant concentration > 3mg/ml).

Add protease inhibitors 0.1 MPMSF 5ul + 5mg/ml Apoprotinin 5ul.

Sonicate for 5 sec x 4 until clear.

Spin down the cell debris at 9000rpm x 15 min by ss34 rotor.

Mix the supernatant with O. lml Ni2+-NTA agarose resin (Qiagen) for 50 min with shaking.

(The resin is equilibrated by 1.5ml buffer F (example 2) for three times before being used. The change of buffer is made by spinning the beads down at 4,000rpm x 1 min microcentrifuge.) Wash three times with 1.5ml of buffer F + 5mM imidazole (Sigma), spin down at 4,000rpm x 1 min.

Elute with 0.25ml of buffer F + 150mM imidazole.

-Incubation for 50 min at 4 degrees C withe gentle mixing.

-Collect the supernatants after centrifugation (14,000rpm x 2 min) Dialyze against buffer A (example 2) (pH 9.0) + 50% glycerol and save in-70 degree C freezer.

Example 4. His tag does not affect virus growth in vivo.

Complementation assay:

A culture of bacterial strain called MF1974, was lysogenized with a lambda vector that had mutations in the terminase enzyme such that the terminase enzyme was rendered ineffective (lambda Aamllam32). The three cultures were subsequently transformed with the following three types of plasmids: lambda vector with functional, unaltered terminase (pJMlD) and lambda vector with a His tag on carboxy terminus of the terminase-encoding gene (pJM1 His6- end).

The relative ratio of complementation (compared to deletion mutant) was 1500 for the functional, unaltered terminase vector as well as for the His tagged terminase lambda vector.

Recombination assay: The series of pJM1 constructs (above) were transformed into a bacterial strain which had been lysogenized with a lambda vector with a mutation in the terminase enzyme, such that the terminase enzyme was rendered inactive (MF2669 with lambda Aam42). Lysates from transformants were made by temperature induction and plated on MF1427 medium. The pJM1 with the histidine tag transformed at a slightly higher efficiency (1.62 x 10 to the 7th pfu/ml) than the pJM1 vector (1.43 x 10 to the 7th pfu/ml). The pJM1 deletion mutant did not transform the cells. This similarity in transformation frequency suggest that terminase with the His

tag on the carboxyl end of the terminase gene (gpA) is fully functional through the packaging process.

Example 5. Retention of cos cleavage activity of His-tagged protein.

Purified His tagged terminase (see example 3) was added to distilled-water-suspended pQH1 (a plasmid with a cos site) and cos cleavage was measured by light spectroscopy (Beckman DU@-64 Spectrophotometer) at OD 595 twice (5ml and 10ml) at the following time periods: 20,40,60,120 and 240 seconds after addition of His-tagged terminase. Linearized pQH1 was subjected to the same conditions. These same procedures were also followed with the exception being that wild type terminase was used. No significant differences between the His tagged terminase cos-cleavaging ability and wild type terminase cos-cleaving activity was detected.

Example 6. Retention of in vitro packaging activity of His-tagged protein.

The following mixture was made to determine the in vitro packaging activity of the His-tagged terminase: 10x DPA 2A1 prohead + tail (crude extract 10 ml at room temperature) DPB + ATP 2, u1 1 DNA cI857 Sam7 2 g)

terminase (His tagged or wildtype) 2) u1 (120 ng/} MesH 2µl (996.5 fil H20+3.5full ß-mercaptoethanol) Packaging occurred at essentially the same frequency for any concentration greater than 30 nM of His-tagged terminase and wild-type terminase.

Although the present invention has been fully described herein, it is to be noted that various changes and modifications are apparent to those skilled in the art.

Such changes and modifications are to be understood as included within the scope of the present invention as defined by the appended claims. INDICATIONS RELATING TO A MICROORGANISM 13bis)(PCTRule A. The indications made below relate to the microorganism referred to in the description on page 1 zinc H. IDENTIFICATION OF DEPOSIT Further deposits are identifie on an additional sh t g Name of depositary institution American Type Culture Collection Address of depositary institution (including postal code and country) Rockville, Maryland US I) ate of depo it | Accession Number 31 October 1997 98562 C. ADDITIONAI INDICATIONS/leavt blank iJnor applicable) This information is continued on an addition shekel 1 ID. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (iftheindicarionsare norrorall d signatedSta J E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable) The indications listed below will be submitted to the Internauonal Bureau later (sntcifythe gokrol nncveofthe indoaonse. g.,"Acression Numlr of Deynosit"} For receiving Office use only For International Bureau use only E This sheet was received with the intemauonal application This sheet was received by the tntcmauona) Burrau nn Authorized officer Authorized officer + *t $ ..