Loading patent…
AssigneeCubist Pharmaceuticals, Inc.
InventorsSusan A. Martinis, Mandana Sassanfar, Sunghoon Kim, Sang Ho Lee, Paul R. Schimmel
Estimating the term…
Isolated and/or recombinant nucleic acids encoding mycobacterial methionyl-tRNA synthetase have been characterized. Recombinant DNA constructs and vectors having a sequence which encodes mycobacterial methionyl-tRNA synthetase have been made, and can be used for the construction of tester strains as well as for the production of isolated and/or recombinant methionyl-tRNA synthetases. These enzymes or portions thereof are useful in the biochemical separation of methionine and quantification of methionine or ATP, and for producing antibodies useful in the purification and study of the enzyme, for example. Host cells and methods useful for producing recombinant mycobacterial methionyl-tRNA synthetases are described, as are tester strains, which are cells engineered to rely on the function of the tRNA synthetase encoded by an introduced cloned gene. Tester strains can be used to identify inhibitors of the essential tRNA synthetase enzyme encoded by the introduced cloned gene, and thus provide a means to assess the antimicrobial effect and specificity of the inhibitor without employing slow-growing, pathogenic strains of mycobacteria, such as Mycobacterium tuberculosis.
An isolated nucleic acid which encodes an aminoacyl-tRNA synthetase of a species of the genus Mycobacterium, wherein said aminoacyl-tRNA synthetase is selected from the group consisting of methionyl-tRNA synthetase, isoleucyl-tRNA synthetase, leucyl-tRNA synthetase, valyl-tRNA synthetase and cysteinyl-tRNA synthetase.
An isolated nucleic acid encoding a methionyl-tRNA synthetase of a species of the genus Mycobacterium.
An essentially pure nucleic acid which encodes a mycobacterial methionyl-tRNA synthetase, wherein said nucleic acid hybridizes to DNA having the sequence SEQ ID NO:b 1, its complement, DNA having the sequence SEQ ID NO:3 or its complement, under conditions comprising a) hybridization overnight in 500 mM NaH.sub.2 PO.sub.4 (H.sub.2 O), 5% dextran sulfate, 7% SDS, 1 mM EDTA, 50% formamide, 250 mM NaCl, and 100 .mu.g/ml salmon sperm DNA at 37.degree. C., b) washing twice in 2.times.SSC, 0.1% SDS for 10 minutes each at room temperature, and c) washing twice in 0.5.times.SSC, 0.1% SDS for one hour each at 65.degree..
An essentially pure nucleic acid which encodes a mycobacterial methionyl-tRNA synthetase, wherein said nucleic acid hybridizes to DNA having the sequence SEQ ID NO:1, its complement, DNA having the sequence SEQ ID NO:3 or its complement, under conditions comprising a) hybridization overnight in 5.times.Denhardt's solution, 5.times.SSC, 0.5% SDS and 2 mM EDTA at 68.degree. C., b) washing twice with 2.times.SSC, 0.1% SDS for one hour at room temperature, and c) washing twice with 0.5.times.SSC, 0.1% SDS for one hour at 65.degree..
An isolated nucleic acid which encodes a methionyl-tRNA synthetase of Mycobacterium tuberculosis.
An essentially pure nucleic acid which encodes a protein comprising a polypeptide having an amino acid sequence corresponding to amino acids 3-521 in SEQ ID NO:2.
A nucleic acid vector comprising nucleic acid encoding an aminoacyl-tRNA synthetase of a species of the genus Mycobacterium, wherein said aminoacyl-tRNA synthetase is selected from the group consisting of methionyl-tRNA synthetase, isoleucyl-tRNA synthetase, leucyl-tRNA synthetase, valyl-tRNA synthetase and cysteinyl-tRNA synthetase.
A nucleic acid vector comprising nucleic acid encoding a mycobacterial methionyl-tRNA synthetase, wherein said nucleic acid hybridizes to DNA having the sequence SEQ ID NO:1, its complement, DNA having the sequence SEQ ID NO:3 or its complement, under conditions comprising a) hybridization overnight in 500 mM NaH.sub.2 PO.sub.4 (H.sub.2 O), 5% dextran sulfate, 7% SDS, 1 mM EDTA, 50% formamide, 250 mM NaCl, and 100 .mu.g/ml salmon sperm DNA at 37.degree. C., b) washing twice in 2.times.SSC, 0.1% SDS for 10 minutes each at room temperature, and c) washing twice in 0.5.times.SSC, 0.1% SDS for one hour each at 65.degree..
A nucleic acid vector comprising nucleic acid encoding a mycobacterial methionyl-tRNA synthetase, wherein said nucleic acid hybridizes to DNA having the sequence SEQ ID NO:1, its complement, DNA having the sequence SEQ ID NO:3 or its complement, under conditions comprising a) hybridization overnight in 5.times.Denhardt's solution, 5.times.SSC, 0.5% SDS and 2 mM EDTA at 68.degree. C., b) washing twice with 2.times.SSC, 0.1% SDS for one hour at room temperature, and c) washing twice with 0.5.times.SSC, 0.1% SDS for one hour at 65.degree..
A recombinant DNA vector comprising DNA which encodes a mycobacterial methionyl-tRNA synthetase.
A host cell comprising a recombinant nucleic acid which encodes an aminoacyl-tRNA synthetase of the genus Mycobacterium, wherein said aminoacyl-tRNA synthetase is selected from the group consisting of methionyl-tRNA synthetase, isoleucyl-tRNA synthetase, leucyl-tRNA synthetase, valyl-tRNA synthetase and cysteinyl-tRNA synthetase.
A host cell comprising a recombinant nucleic acid, wherein said nucleic acid encodes a protein comprising a mycobacterial methionyl-tRNA synthetase.
A host cell comprising a recombinant nucleic acid, wherein said nucleic acid encodes a protein comprising a polypeptide having an amino acid sequence corresponding to amino acids 3-521 in SEQ ID NO:2.
A host cell which contains a recombinant mycobacterial methionyl-tRNA synthetase gene which can express a mycobacterial methionyl-tRNA synthetase.
An expression vector comprising a nucleic acid encoding a fusion protein comprising a mycobacterial aminoacyl-tRNA synthetase, wherein said aminoacyl-tRNA synthetase is selected from the group consisting of methionyl-tRNA synthetase, isoleucyl-tRNA synthetase, leucyl-tRNA synthetase, valyl-tRNA synthetase and cysteinyl-tRNA synthetase, and wherein the nucleic acid is under the control of transcriptional signals and is linked to appropriate translational signals for expression of said fusion protein in a suitable host cell.
A method for producing active mycobacterial methionyl-tRNA synthetase comprising the following steps:
A method for producing a mycobacterial aminoacyl-tRNA synthetase selected from the group consisting of methionyl-tRNA synthetase, isoleucyl-tRNA synthetase, leucyl-tRNA synthetase, valyl-tRNA synthetase and cysteinyl-tRNA synthetase, comprising maintaining a host cell comprising a recombinant nucleic acid encoding said mycobacterial aminoacyl-tRNA synthetase under conditions suitable for expression of the nucleic acid, whereby the encoded mycobacterial aminoacyl-tRNA synthetase is expressed and thereby produced.
A method for producing a protein comprising a mycobacterial methionyl-tRNA synthetase, comprising maintaining a host cell comprising a recombinant nucleic acid encoding said protein under conditions suitable for expression of the nucleic acid, whereby the encoded protein is expressed and thereby produced,
A method for producing a protein comprising a mycobacterial methionyl-tRNA synthetase, comprising maintaining a host cell comprising a recombinant nucleic acid encoding said protein under conditions suitable for expression of the nucleic acid, whereby the encoded protein is expressed and thereby produced,
A method for producing a protein comprising a methionyl-tRNA synthetase of a species of the genus Mycobacterium, comprising maintaining a host cell comprising a recombinant nucleic acid encoding said protein under conditions suitable for expression of the nucleic acid, whereby the encoded protein is expressed and thereby produced.
A method for producing a protein comprising a methionyl-tRNA synthetase of M. tuberculosis, comprising maintaining a host cell containing a recombinant nucleic acid encoding said protein under conditions suitable for expression of the nucleic acid, whereby the encoded protein is expressed and thereby produced.
An isolated nucleic acid comprising a nucleic acid which hybridizes to a DNA strand having the sequence shown in SEQ ID NO:1 or an RNA counterpart thereof under conditions comprising a) hybridization overnight in 500 mM NaH.sub.2 PO.sub.4 (H.sub.2 O), 5% dextran sulfate, 7% SDS, 1 MM EDTA, 50% formamide, 250 mM NaCl, and 100 .mu.g/ml salmon sperm DNA at 37.degree. C., b) washing twice in 2.times.SSC, 0.1% SDS for 10 minutes each at room temperature, and c) washing twice in 0.5.times.SSC, 0.1% SDS for one hour each at 65.degree..
An isolated nucleic acid, wherein said nucleic acid encodes a protein comprising a methionyl-tRNA synthetase which is encoded by pSH101A as deposited under ATCC Accession No. 98376.
A host cell comprising a recombinant nucleic acid, wherein said nucleic acid encodes a protein comprising a methionyl-tRNA synthetase which is encoded by pSH101A as deposited under ATCC Accession No. 98376.
Plasmid pSH101A which is deposited in host cells under ATCC Accession No. 98376.
Dependencies are inferred from the claim text.