Production of CPD glycosylases
AssigneeApplied Genetics Incorporated Dermatics
InventorsDaniel B. Yarosh, Leonard F. Estis, Eliyahu Kraus
Life of this patent
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Abstract
Recombinant forms of DNA sequences for CPD glycosylases, including the bacteriophage T4 gene denV, are described that are capable of expression at high levels. Active CPD glycosylases can be recovered from inclusion bodies resulting from the high expression using, for example, a homogenization process which employs stream mixing, and the active proteins can be used in, for example, topical formulations for treatment of photosensitive diseases. Stream mixing can also be used to solubilize inclusion bodies containing proteins other than CPD glycosylases.
Claims16
An isolated, synthetic, and/or recombinant polynucleotide comprising:
- (a) a nucleotide sequence encoding a native amino acid sequence of a cyclobutane pyrimidine dimer (CPD) glycosylase protein from an organism selected from the group consisting of Bacteriophage T4, Bacteriophage RB69, Paramecium bursaria Chlorella virus I, Prochlorus marinus, Bordetella parapertussis, Haemophilus ducreyi, Brucella melitensis, Pasteurella multocida, Micrococcus luteus, Nitrosomonas europeaea and Azoarcus sp. EbN1;
- or
- (b) a complement of said nucleotide sequence, wherein the complement and the nucleotide sequence consist of the same number of nucleotides and are 100% complementary;
- wherein:
- (I) Groups A and B represent the following codons for the following amino acids:
- Amino Acid
- Codon
- Group A
- Tyrosine
- TAC
- Glycine
- GGT
- Valine
- GTT
- Serine
- TCT
- Alanine
- GCT or GCA
- Group B
- Isoleucine
- ATC
- Phenylalanine
- TTC
- Aspartic Acid
- GAC
- Histidine
- CAC
- and
- (II) the nucleotide sequence comprises:
- (a) at least two codons from Group A for more than 45% of the occurrences of the amino acid coded by the codon in the CPD glycosylase protein and at least two codons from Group B for more than 45% of the occurrences of the amino acid coded by the codon in the CPD glycosylase protein;
- or
- (b) at least three codons from Group A for more than 35% of the occurrences of the amino acid coded by the codon in the CPD glycosylase protein and at least three codons from Group B for more than 35% of the occurrences of the amino acid coded by the codon in the CPD glycosylase protein.
An isolated, synthetic, or recombinant polynucleotide comprising:
- (a) a nucleotide sequence encoding a native amino acid sequence of a cyclobutane pyrimidine dimer (CPD) glycosylase protein from an organism selected from the group consisting of Bacteriophage T4, Bacteriophage RB69, Paramecium bursaria Chlorella virus I, Prochlorus marinus, Bordetella parapertussis, Haemophilus ducrevi, Brucella melitensis, Pasteurella multocida, Micrococcus luteus, Nitrosomonas europeaea and Azoarcus sp. EbN1;
- or
- (b) a complement of said nucleotide sequence, wherein the complement and the nucleotide sequence consist of the same number of nucleotides and are 100% complementary;
- wherein the nucleotide sequence comprises the following codons for more than 75% of the occurrences of the amino acid coded by the codon in the CPD glycosylase protein:
- Amino Acid
- Codon
- Tyrosine
- TAC
- Glycine
- GGT
- Valine
- GTT
- Serine
- TCT
- Alanine
- GCT or GCA
- Isoleucine
- ATC
- Phenylalanine
- TTC
- Aspartic Acid
- GAC
- Histidine
- CAC.
An isolated, synthetic, or recombinant polynucleotide comprising the nucleotide sequence of SEQ ID NO:
- 1 or a complement of the nucleotide sequence, wherein the complement and the nucleotide sequence consist of the same number of nucleotides and are 100% complementary.
An isolated, synthetic, or recombinant polynucleotide comprising:
- (a) a nucleotide sequence encoding a T4 endonuclease V protein having the amino acid sequence of SEQ ID NO:
- 2;
- or
- (b) a complement of the nucleolide sequence, wherein the complement and the nucleotide sequence consist of the same number of nucleotides and are 100% complementary;
- wherein:
- (I) Groups A and B represent the following codons for the following amino acids:
- Amino Acid
- Codon
- Group A
- Tyrosine
- TAC
- Glycine
- GGT
- Valine
- GTT
- Serine
- TCT
- Alanine
- GCT or GCA
- Group B
- Isoleucine
- ATC
- Phenylalanine
- TTC
- Aspartic Acid
- GAC
- Histidine
- CAC
- and
- (II) the nucleotide sequence comprises:
- (a) at least two codons from Group A for more than 45% of the occurrences of the amino acid coded by the codon in the T4 endonuclease V protein and at least two codons from Group B for more than 45% of the occurrences of the amino acid coded by the codon in the T4 endonuclease V protein;
- or
- (b) at least three codons from Group A for more than 35% of the occurrences of the amino acid coded by the codon in the T4 endonuclease V protein and at least three codons from Group B for more than 35% of the occurrences of the amino acid coded by the codon in the T4 endonuclease V protein.
Dependencies are inferred from the claim text.