Uracil-DNA nuclease: protein enzyme possessing nuclease activity specific for uracil containing nucleic acid, process for its preparation and methods of use

Information

  • Patent Application
  • 20080032377
  • Publication Number
    20080032377
  • Date Filed
    June 07, 2005
    19 years ago
  • Date Published
    February 07, 2008
    16 years ago
Abstract
A uracil-specific endonuclease enzyme is discovered in specific developmental stages of Drosophila melanogaster. The protein responsible for this enzymatic activity has been isolated, cloned, sequenced, and expressed to practical homogeneity. Sequence homologues have been identified from four additonal organisms. The activity of this enzyme is strictly specific for uracil-containing DNA and persists in the presence of 1 mM concentration of divalent metal ion chelating agent ethylenediaminetetraacetic acid. The unique specificity and the observed metal-ion independent characteristics of this enzymatic activity allow its use for specific degradation of uracil-DNA in a simple enzymatic process while normal DNA is uncleaved by the enzyme. Claims of the present invention concern the enzyme, its homologues, its production, as well as its use for specific degradation of uracil-DNA in any application it might occur, all based on the discovery of the novel uracil-specific nuclease enzyme.
Description

The coding sequence of this protein was found in Drosophila melanogaster samples of different developmental stages as transcribed into mature messenger RNA by reverse transcription of the total mRNA pool into cDNA followed polymerase chain reaction with adequate primer oligonucleotides. The cDNA coding sequence corresponding to SEQ ID NO:6 was cloned into expression vectors as follows:


It was subcloned into vector pET22b (Novagen, EMD Biosciences, Darmstadt, Germany), using EcoRI-Xhol restriction sites originating in plasmid PETUDE. The coding sequence for a maltose binding protein was excised from plasmid pMal-c2E (New England BioLabs, Schwalbach, Germany) by Ndel-BamHI digestion, and was subcloned into plasmid PETUDE using Ndel-BamHI restriction sites originating in plasmid pETMalUDE. In plasmid pETMalUDE, the coding sequence of uracil-DNA endonuclease is at the 3′-end to the coding sequence of the maltose-binding protein. The nucleic acid segment encoding the uracil-DNA endonuclease coding sequence was also subcloned into vector PET19b (Stratagene), using Ndel-Xhol restriction enzyme sites originating in plasmid pETHisUDE. In pETHisUDE, the coding sequence of uracil-DNA endonuclease is at the 3′-end to the coding sequence of the ten-histidine affinity tag.


The expression vectors pETMalUDE and pETHisUDE were used to transform E. coli cells. Expression of the uracil-DNA nuclease protein with the affinity tag maltose binding protein or polyhistidine was performed by iso-propyl-thio-galactoside induction following general methods known to one skilled in the art. It will be evident to one skilled in the art that similar procedures without the exercise of inventive skill may easily result in expression vectors also containing the coding sequence of uracil-DNA nuclease. either with an affinity tag or without such tag. Such vectors may also be used for expression of the protein uracil-DNA nuclease using methods known in the art. Such equivalents are intended to be encompassed by the following claims.


Due to the known degeneracy of the genetic code, it will be also apparent to those skilled in the art that different, but equivalent nucleotide sequences which code for the uracil-DNA nuclease enzyme of the invention, as shown in SEQ ID NO:1, or SEQ ID NO:2, or SEQ ID NO:3, or SEQ ID NO:4, or SEQ ID NO:5, may be isolated, synthesized or otherwise prepared without the exercise of the inventive skill. Such degenerate and equivalent coding sequences are included within the scope of the present invention.


Uracil-DNA nuclease activity of the recombinant uracil-DNA nuclease protein. The protein was expressed as described above. It was isolated from E. coli cell lysate and purified using the affinity tag with usual methods known in the art. Its activity was tested on uracil-DNA and normal DNA. Uracil-DNA was produced in the form of a plasmid isolated from dut-ung-double mutant E. coli cells. Normal DNA was produced in the form of plasmid from wild type E. coli cells. Plasmid preparations were according to usual methods known in the art. The recombinant protein, purified to approximataly 98% homogeneity, and containing the amino acid sequence constituted in SEQ ID NO:1 was incubated with normal DNA and uracil-DNA at 37.degree.C. for 10, 30, and 60 minutes in a solution containing 10 micrograms/ml uracil-DNA nuclease, 20 micrograms/ml DNA, 25 mmole/liter Hepes buffer, 150 mmole/liter sodium-chloride. Uracil-DNA was fragmented into small oligonucleotides while normal DNA showed practically no fragmentation at all. It was concluded that the protein enzyme uracil-DNA nuclease is strictly specific for uracil-DNA and is capable of fragmenting it into smaller oligonucleotides.


DNA binding affinity of recombinant uracil-DNA nuclease. The DNA binding affinity was tested on gel shift assay using usual methods known in the art. The recombinant uracil-DNA nuclease was shown to bind to uracil-DNA and normal DNA with comparable affinities. It was concluded that uracil-DNA nuclease has a general DNA binding ability which is not strictly specific for uracil-DNA.


Homologues of uracil-DNA nuclease. Homologue sequences of uracil-DNA nuclease from Drosophila melanogaster as contained in the amino acid sequence constituted in SEQ ID NO:1 or as contained in the nucleotide sequence constituted in SEQ ID No:6 were used to search for homologues in the usual databases known in the art. Four such homologues were identified, as contained in the amino acid sequence constituted in SEQ ID NO:2, or SEQ ID NO:3, or SEQ ID NO:4, SEQ ID NO:5, or as contained in the nucleotide sequence constituted in SEQ ID No:7, or SEQ ID No:8, or SEQ ID No:9, or SEQ ID No:10. All these homologues are present in genomes of metamorphing insects. It is concluded that the sequences contained in amino acid sequences constituted in SEQ ID NO:1, or SEQ ID NO:2, or SEQ ID NO:3, or SEQ ID NO:4, SEQ ID NO:5. correspond to proteins specific to metamorphing insects and other organisms may only encode such distant relatives that are not evident at the present.


Novelty of the uracil-DNA nuclease enzyme protein. The protein as contained in the amino acid sequence constituted in SEQ ID NO:1, or SEQ ID NO:2, or SEQ ID NO:3, or SEQ ID NO:4, SEQ ID NO:5. does not show significant homology to any of the known nucleases. Its active site may therefore constitute novel characteristics, exploitable in molecular biology application. Such an example may be that the enzymatic activity of uracil-DNA nuclease protein which is the subject of the present invention does not require the presence of divalent metal ions. This characteristics makes this enzyme a useful and rather unique tool for any molecular biology, or other applications where divalent metal ion-independent nuclease activity is used. Such applications are intended to be encompassed in the present invention.


Methods of use of uracil-DNA nuclease. The uracil-DNA nuclease of the present invention may be used in any circumstances where specific degradation of uracil-DNA is required. Such applications that require the protein enzyme uracil-DNA nuclease as contained in the amino acid sequence constituted in SEQ ID NO:1, or SEQ ID NO:2, or SEQ ID NO:3, or SEQ ID NO:4, SEQ ID NO:5. are intended to be encompassed in the present invention. Due to the equivalent base-pairing capabilities of uracil and thymine, uracil-DNA and normal DNA may encode equivalent genetic information, if uracil is present only at thymine-replacing sites. One example for a useful application of uracil-DNA nuclease concerns biosafety.


Biosafety application of uracil-DNA nuclease. For specific degradation of recombinant DNA encoding potentially not desired, or even harmful, genetic information produced in vitro under laboratory circumstances, the use of uracil-DNA nuclease provides a rather simple and straightforward solution. Recombinant DNA is frequently produced in the laboratory and its escape from the laboratory is not always strictly ensured. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, it will be possible, using general methods known in the art, to produce all recombinant DNA with a significant uracil content. For in vitro production, inclusion of dUTP into the polymerase reactions will ensure the production of uracil-DNA. For cellular experiments in E. coli, the use of dut-ung-mutant E. coli strain will ensure the production of uracil-DNA. These uracil-DNA species will be equivalent in genetic coding to normal DNA of the same sequence where the deoxyuridine residue is replaced by thymine. Recombinant DNA with uracil content will be degradable by uracil-DNA nuclease with high efficiency, while normal DNA will not be degraded. Recombinant DNA will be therefore prevented from escaping the laboratory by the use of uracil-DNA nuclease.


Molecular diagnostics application of uracil-DNA nuclease. To ascertain the presence of a specific mutation in e.g. human or other genome, uracil-DNA nuclease may be applied. Some defined mutations in the human genome are well known to be involved in several pathogenic conditions. Molecular diagnostics techniques are known in the art to recognize such mutations by sequencing. With the use of uracil-DNA nuclease, a simple method with ease of use may be designed that does not require sequencing. In such a method, DNA oligomers hybridizing to the mutated site may be synthesized containing deoxyuridine residues. The oligomers may be labelled at the 5′ and 3′ end, with flurescent dyes capable of flurescence energy transfer. The labelling may be designed in such a way that within the intact oligomer, fluorescence is quenched due to the short distance between the two fluorescent labels as defined by the length of the oligonucleotide. However, fluorescence may be significantly increased if the distance between the two labels increase due to cleavage of the oligonucleotide. Such a deoxyuridine residue-containing double-labelled oligonucletide with its controlled sequence that is 100% complementary to the sequence containing the mutation to be investigated may hybridize to a site if it contains the mutation to be investigated. If the mutation is not present, hybridization will not occur. After the hydridization experiment, the hybrid double stranded DNA may be separated from single stranded DNA by usual methods known in the art. The double stranded DNA may then be treated with uracil-DNA nuclease that will result in cleavage of the uracil-containing oligonucleotide strand and therefore fluorescence intensity will increase. Increment in flurescent intensity, as detected by methods known in the art, will then reflect the existence of the mutation.


Persons skilled in the art will recognize, or will be able to design using no more than routine experimentation, many equivalents of the specific embodiments of the invention described herein. Such equivalents are intended to be included in the present invention.










SEQ ID NO:1









M I K C H M P S S W R R L R K I S R I L A L T G S






R Q I L T Q V L A T K G A A M A E G D S





K F G F K D M E K A L E T L K L L E S H





D M Q Y R K L T V R G L L G R A K R V L





T M T K A E E K L K N I N A A I G V F E





K W L E E N G G G A S S K N A K T E S E





D K V E T V P G L G F K D K A A A E A T





L S I L A E R D P D Y Q R L A I K G L I





G S S K R V L S G T K N E D K I T A I K





E G V Q V L E D F L E K F E A E N R I K





D N R A Y L P L A V V T K L P D P K D E





L A K E F L E A Y G G S K A K G N Y K H





L R T M F P K T D E K T S W D I V R N R





Q L S K L L E Q I K S E E A K L F D A E





T G A P T D L H L Q L I H W A Y S P Q P





D K L K Q Y I E K L A K K T P E K R K Q





E S S S S A S D S S A T S Q D S D G E D





K P K R K K K R E E











SEQ ID NO:2









M S E G E S K F G F K D M E K A L E T L






K L L E D H D M Q Y R K L T V R G L L G





R A K R V L T L T K A E E K L K N I N E





A I G V F E K W L E D N G G G A S S K N





A K T D N E D K V E T V P G L G F K D K





A A A E A T L S I L A E R D P D Y Q R L





A I K G L I G S S K R V L S G T K N E D





K I N S I K E G V Q V L E D F L E K F E





A E N R I K E N R A Y L A Y A V V S K L





P E P K E D L S V E F L A A Y G G S K A





K G N Y K H L R T M Y P K E D D T T S W





D I V R N R Q I A K L L E Q I K S E E A





K L F D S E S G E P T D L H L Q L I H W





A Y T P Q P D K V K A Y V E K L A K K T





P Q K R K P E











SEQ ID NO:3









M A K E E S K Y G F K D K A






K A E E S L E L L K S E D H K Y Q L L T





V R G L I G R A K R V L T L T K A E D K





I N N I K A A I E T F E Q W L E A N S S





S S T K N A K P K D A E D K V E T V P G





L G F K D K Q A A E Q T L S I L E G R D





P D Y Q K L A I K G L I G S A K R V I P





A T K N E E K L S S I K Q A V A L F E D





F L D R F D R E E R G K Q N M P Y L S I





D L I R Q L P A P Q G E Q S D K L A V E





F L A C Y E T Q A K G N Y K H L R T K A





P K D P G S K T W D I V R N A K L Q A L





K P D S S V K L F D Q D G K P T E L H L





Q M V Q W A Y S P Q V E K L K S Y A N S





L A S S G K S T T P S R K R T H S S S S





S S E Q E S K A K E S K K D R K K S K K











SEQ ID NO:4









K S T E P E E S V M G F K D K Q K A L D






T L K A L D G R D I S Y Q Y H V I A S F





V S R A K R T L Q I T R D E E K L A N I





R E A L K V F E D W L A N Y K E N N R S





K E N L A Y L P I E T I K G F K S L A K





N G L G F K D K E K A L Q T I K L L E G





R D L N Y Q Y H A I S G L V K R A E R V





I S C T K D E Q K L K N I K E A V E V F





D N W I T D F K V N G R A K M N F D Y L





S V D L V R S Y K P L A D K Y K I E D N





G F L K A Y E E V D G D Y K K L R N V Q





V P D S S I T W D I E R N K N L Q N V V





D R V K E Q K K W F E T D G E F E D L P





T E G H I R C I M W A Y S H D A G K L K





K L L P T L A E K L K S











SEQ ID NO:5









M L Y K N E R S V K L T I G R V D S D N






R Q K F S D R P I A Q Y A S D F N V E V





F I V L C F R T M G K D D K E D T G F G





F K D K A K A E D T L R L L E E H D L N





Y R R L T V R G L L G R A K R V L S M T





K A E E K I K N I K E A M E V F E N W L





A D L D K N K E Q K E K P E K K E K K D





T V P G L G F K D K S A A E G T L K V L





D G R D P D Y Q R L A V K G L I G R A K





R V L T C T R D E T K V S N I K E A I T





V F E K F L D D F E S L H L S K E N N P





Y L S L G V V R A A E Q L A G E S K S F





I A A Y S S V N G E Y K R L R T V E E S





E G G L T W D I V R N N A L K P L K A T





H A E A K L F N E E G E P T P E H L E L





I L W A Y S P E A A R L K K C L P E V E





T E V S R K R R S S A Q E E S P A K K K





K D











SEQ ID NO:6









atgattaagtgccatatgccgtcgagttggagacggctacgcaaaatcag






tcgtattctagcgctgacaggaagcagacagatactcacccaagtattag





caaccaaaggagcagcaatggcagagggagattcgaaatttggcttcaag





gacatggagaaggcgttagagacgttgaagctgctggagagtcacgacat





gcagtatcgcaagctgacggtgcgcggtttgcttggccgggccaaaagag





tcctgacaatgaccaaggcggaggagaagctgaagaacatcaatgcggcc





attggagtctttgaaaagtggctggaggagaatggcggaggggcgtccag





caagaatgccaagacagagagcgaggacaaggtggagacggtgccgggat





tgggattcaaggacaaggctgctgcggaggcaacgctgagcattttggcg





gaacgagatccggactaccagaggttggccatcaagggattgattggcag





ctccaagcgtgtcctgtcaggcaccaagaacgaggacaagatcacggcca





taaaggagggagtccaggtacttgaggatttcctcgaaaagttcgaggcc





gagaatcgtatcaaggacaatcgagcatacttgccactcgccgtggtcac





caaactgcccgatcccaaagatgagttggctaaggagtttctcgaagcct





atggcggctccaaggccaagggtaactacaagcacctgcgcacaatgttc





cccaaaacggatgaaaagaccagctgggatattgtgcgcaatcgtcagct





gtccaagttgctggagcagattaagagtgaggaggccaagctcttcgatg





cagagaccggagcacccaccgacctgcacctgcagttgatccactgggca





tacagtccgcagccggacaagctgaagcagtacatcgaaaagctggccaa





gaagacgcccgaaaagcgcaagcaggagagcagcagcagtgccagcgatt





ccagtgccaccagccaggattccgatggcgaggataagcccaaaaggaag





aagaagagggaggag











SEQ ID NO:7









atgtcggagggagagtcaaagtttggtttcaaggacatggagaaggccct






ggagacgctgaagctgctggaggatcatgacatgcagtaccgaaagctga





ccgtgcgcggtctccttggacgcgccaagcgagtgctgaccttgaccaag





gcggaggagaagctgaagaacatcaatgaggcgattggcgtgttcgagaa





atggctggaggataatggcggcggggcgtccagcaagaacgccaagactg





acaacgaggataaggtggagaccgtgcccggactgggcttcaaggacaag





gcggcggcggaggcgacgctgagcattctggcggagcgtgacccggacta





ccagaggctggccatcaagggattgattggcagctccaagcgagtgctgt





ccggcaccaagaacgaggacaagatcaattccatcaaggagggagtccag





gtgctggaggatttcctggagaagttcgaggccgagaaccgcatcaagga





gaatcgcgcctacttggcctatgccgtcgtgtccaagctgccagagccca





aagaggatctgtccgtcgagttcctggctgcctacggcggctccaaggcc





aagggcaactacaagcacctgcgcaccatgtaccccaaggaggacgacac





caccagctgggacattgtgcgcaatcgccagatagccaagctgctggagc





agatcaagagcgaggaggccaagctgttcgactcggagtcgggcgagccc





acagatctccacttgcagctgatccactgggcctacacgccccagccgga





caaggtgaaggcctatgtggagaagctggccaagaagacgccgcagaagc





gcaagccggag











SEQ ID NO:8









atggcaaaggaagaatcaaagtacggcttcaaggataaggcc






aaggccgaggagtcgctggagctgctgaagagcgaggatcacaagtacca





gctgctgacggtgcgcggtctgatcggacgggcgaagcgtgtgctgacat





tgacgaaggctgaggacaagataaacaacataaaggctgcgatcgaaacg





ttcgagcagtggctggaagcaaacagctcctccagcaccaaaaacgcaaa





gccgaaggatgcagaagacaaggtggaaactgtgccagggttgggtttca





aggacaagcaggcggctgaacaaacgctgagcatcctagaagggcgcgat





cccgattatcagaagctagcaatcaagggactgatcggtagcgcaaagcg





cgttatccctgccaccaagaacgaggagaagctaagctcgatcaagcaag





cggtggcactgtttgaagactttctcgatcggttcgatcgcgaggagcgg





ggcaagcaaaacatgccgtacctttcgatcgacttgatacgtcaactgcc





cgcaccgcagggggagcagtcggacaagctggcagtggaatttctcgcct





gctatgaaacgcaggccaaaggcaactacaaacatttgcgcaccaaagca





cccaaggacccaggctcgaagacgtgggacattgtgcgaaatgcgaaact





gcaggcactgaaaccggacagcagtgtgaagttattcgatcaggacggca





agcctaccgagctgcacttgcagatggtacagtgggcgtacagcccacag





gtggagaagctgaagagctatgcgaacagtttggcgagcagtggcaaatc





aacaacaccgtccaggaaacgaacgcactcttccagctcatcttcggagc





aggagtcaaaggcgaaggagagtaagaaggatcgcaaaaagtcgaagaaa











SEQ ID NO:9









aaatcaacggaaccggaagaatccgtgatgggtttcaaggataagcaaaa






ggccttggacacgctgaaagctctggacggccgagatatcagttatcagt





accatgtgattgctagttttgtgagccgcgcgaaaagaacgttgcagatt





acaagggacgaggaaaagctggccaatatacgagaagctttgaaagtgtt





cgaggattggctcgctaactacaaagagaacaatcgcagtaaagaaaacc





tcgcttacttgccaatcgagactataaaaggcttcaagagccttgccaaa





aacggactcggttttaaagataaagagaaggctttgcagactatcaagtt





actggaaggtagggatctaaattatcagtaccacgcgatctctggccttg





tgaaaagagctgagcgagtgatatcgtgcacaaaggacgagcaaaaactc





aagaacataaaagaagctgtggaagtgttcgacaattggattacggattt





caaggtaaatggccgggcaaagatgaatttcgattatttatccgttgatt





tggtacgatcctacaaaccgttggcggacaagtacaaaatcgaagataat





ggatttcttaaagcgtacgaagaagtggatggagattataagaaattgag





aaacgttcaagttccagattcgagtattacctgggatatagagaggaata





agaatcttcaaaatgtcgtagatcgtgtcaaagaacaaaagaaatggttc





gagacggatggtgagttcgaagatttacccaccgaaggacacattcgatg





tataatgtgggcttacagtcacgatgcaggtaaattgaaaaaacttttgc





ctacgttagctgaaaagttaaaatcg











SEQ ID NO:10









atgttatataaaaacgaaagatctgtcaagttaactataggtagagttga






ttcagacaatcgtcaaaaattctctgatcgtccgatagcacagtatgcat





ctgactttaatgtggaagtttttattgttttgtgtttcagaacaatgggc





aaagatgataaagaagatacaggatttggattcaaggacaaggcgaaggc





ggaggacacgctgcggctcctggaggagcacgacctgaactacaggaggc





tgacagtgagaggacttctcggtagagcgaagagggttctgtcaatgacg





aaagcagaagagaaaatcaaaaacatcaaagaggccatggaggtgttcga





gaactggctcgcggacctcgacaagaacaaggagcaaaaagagaagcccg





aaaagaaagagaagaaagacactgtgccgggcctaggcttcaaggataag





tctgccgcagaagggaccctcaaagtgttggacgggagagacccggatta





ccagagactggccgtcaagggccttatagggagagccaagcgggtgttga





cttgcacccgagatgagactaaagtatcgaacatcaaggaggccataacg





gtcttcgagaagttcctcgacgacttcgagagcttgcatctgagcaaaga





gaacaacccgtacctaagtctcggcgtggtgcgggccgccgagcagctgg





ccggggagagcaaatcgttcatagccgcctactcctccgtcaatggagaa





tacaagaggctgaggaccgtggaggagtcggaggggggcctcacttggga





catcgttaggaacaacgcgctcaaaccgctcaaagccacgcatgctgagg





cgaagctgttcaacgaagaaggcgaaccgaccccggaacatttagagtta





atactgtgggcgtactcaccggaggcggcccgcctcaagaagtgccttcc





cgaggtggaaacagaagttagccggaagagaagaagcagcgcgcaagaag





agtctccggctaaaaagaagaaggat





Claims
  • 1. An isolated polypeptide comprising an amino acid sequence which shows at least 90% identity to any of the amino acid sequences selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5.
  • 2. The isolated polypeptide of claim 1, wherein the polypeptide is a polypeptide present in any of the following organisms: Drosophila melanogaster, or Drosophila pseudoobscura, or Anapheles gambiae, or Apis mellifera, or Bombyx mori.
  • 3. An isolated nucleic acid sequence which shows at least 90% identity to any of the nucleic acid sequences selected from the group consisting of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10.
  • 4. A recombinant vector containing a nucleic acid sequence according to claim 3.
  • 5. The recombinant vector of claim 4 which is an expression vector.
  • 6. The recombinant vector of claim 5 wherein the nucleic acid sequence is linked to a second nucleic acid sequence encoding a heterologous amino acid sequence.
  • 7. An isolated polypeptide comprising at least 55 consecutive amino acid residues of SEQ ID NO:1 and which has at least one bioactivity of the uracil-DNA nuclease enzyme; wherein the bioactivity is selected from the group consisting of: (a) binding to DNA(b) cleaving uracil-substituted DNA, wherein thymine bases are replaced by uracil bases in any given sequence constraint, but not cleaving normal DNA.
  • 8. A recombinant vector containing a nucleic acid sequence encoding the amino acid sequence according to claim 7.
  • 9. The recombinant vector of claim 8 which is an expression vector.
  • 10. The recombinant vector of claim 9 wherein the nucleic acid sequence is linked to a second nucleic acid sequence encoding a heterologous amino acid sequence.
  • 11. The recombinant vector of claim 5 wherein the heterologous amino acid sequence is an affinity purification tag sequence or a secretion signal sequence.
  • 12. A host cell transformed with the recombinant vector of claim 4.
  • 13. The transformed host cell of claim 12 which is an E. coli host cell.
  • 14. A host cell transformed with the recombinant vector of claim 5.
  • 15. The transformed host cell of claim 14 which is an E. coli host cell.
  • 16. A process for obtaining a recombinant uracil-DNA nuclease enzyme protein, the process comprising the following steps: (a) culturing the transformed host cell of claim 12 in culture medium under conditions inducing expression of the recombinant uracil-DNA nuclease by the transformed host cell; (b) lysing host cells to produce a cell lysate comprising the recombinant uracil-DNA nuclease enzyme protein and other materials; (c) performing chromatography on an affinity column corresponding to an affinity purification tag present on the recombinant uracil-DNA nuclease enzyme protein to obtain a fraction enriched in the uracil-DNA nuclease enzyme protein; (d) performing size exclusion chromatography to obtain the purified uracil-DNA enzyme protein.
  • 17. The process of claim 16 wherein the affinity tag is maltose binding protein or a polyhistidine peptide.
  • 18. The process of claim 16 wherein the chromatographic step in claim 16 is replaced by ion exchange chromatography.
  • 19. A process for specific cleavage of uracil-DNA by the enzyme protein of amino acid sequence according to claim 1.
  • 20. A process for specific cleavage of uracil-DNA by the enzyme protein comprising the amino acid sequence according to claim 7.
Provisional Applications (1)
Number Date Country
60521645 Jun 2004 US