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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/93—Ligases (6)
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
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  • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
  • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/67—General methods for enhancing the expression
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P21/00—Preparation of peptides or proteins
  • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

Definitions

• Kits are also a feature of the invention.
• a kit for producing a protein with a brominated or iodinated amino acid at a specified position is provided, where the kit includes a cell comprising an orthogonal tRNA that functions in the cell and recognizes a selector codon and an orthogonal aminoacyl-tRNA synthetase, packaged in one or more containers.
• Panel B illustrates a portion of the crystal structure of the B. stearothermophilus TyrRS- tyrosyl adenylate complex.
• the five residues of M. jannaschii TyrRS which were randomized are in parenthesis (i.e., Tyr 32 , Glu 107 , Asp 158 , lie 159 and Leu 162 ). Residues were selected based on observed contacts between the homologous B. stearothermophilus TyrRS residues (Tyr , Asn , Asp , Phe and Leu ) and tyrosyl adenylate in the crystal structure.
• the relative ratio of O-tRNA charged by the O-RS to endogenous tRNA charged by the O-RS is high, preferably resulting in the O-RS charging the O-tRNA exclusively, or nearly exclusively, when the O-tRNA and endogenous tRNA are present in equal molar concentrations in the translation system.
• Selection or screening agent refers to an agent that, when present, allows for selection/screening of certain components from a population.
• a selection or screening agent can be, but is not limited to, e.g., a nutrient, an antibiotic, a wavelength of light, an antibody, an expressed polynucleotide, or the like.
• the selection agent can be varied, e.g., by concentration, intensity, etc.
• RS can further include an O-tRNA, where the O-RS preferentially aminoacylates the O- tRNA with an unnatural amino acid such as a brominated or iodinated amino acid (e.g., bromoPhe or iodoPhe).
• a composition including an O-RS can further include a translation system (e.g., in vitro or in vivo).
• a nucleic acid that comprises a polynucleotide that encodes a polypeptide of interest, where the polynucleotide comprises a selector codon that is recognized by the O-tRNA, or a combination of one or more of these can also be present in the translation system.
• An unnatural amino acid is typically any structure having Formula I wherein the R group is any substituent other than one used in the twenty natural amino acids. See e.g.. Biochemistry by L. Stryer, 3 rd ed. 1988, Freeman and Company, New York, for structures of the twenty natural amino acids. Note that the unnatural amino acids of the invention can be naturally occurring compounds other than the twenty alpha-amino acids above (or, of course, can be artificially produced synthetic compounds).
• Synthetech, Inc. (on the world wide web at synthetech.com) and Advanced Asymmetries, hie. (advancedasymmetrics.com).
• Those that are not commercially available are optionally synthesized as provided in various publications or using standard methods known to those of skill in the art.
• organic synthesis techniques see, e.g., Organic Chemistry by Fessendon and Fessendon, (1982, Second Edition, Willard Grant Press, Boston Mass.); Advanced Organic Chemistry by March (Third Edition, 1985, Wiley and Sons, New York); and Advanced Organic Chemistry by Carey and Sundberg (Third Edition, Parts A and B, 1990, Plenum Press, New York).
• non-stochastic mutagenesis which uses polynucleotide reassembly and site-saturation mutagenesis can be used to produce enzymes and/or pathway components, which can then be screened for an ability to perform one or more synthetase or biosynthetic pathway function (e.g., for the production of unnatural amino acids in vivo). See, e.g., Short “Non-Stochastic Generation of Genetic Vaccines and Enzymes" WO 00/46344.
• the O-RS comprises an amino acid sequence encoded by a polynucleotide sequence comprising SEQ ID NO:l or 2, or a complement thereof, hi certain embodiments of the invention, the O-RS preferentially aminoacylates the O-tRNA with the brominated or iodinated amino acid with an efficiency of at least 50% (e.g., at least 60%, at least 75%, at least 80%, or at least 90% or more) of the efficiency with which a polypeptide comprising an amino acid sequence of SEQ ID NO: 3 or 4 preferentially aminoacylates the O-tRNA with the brominated or iodinated amino acid.
• an O-tRNA of the invention comprises or is encoded by a polynucleotide sequence as set forth in the sequence listing or examples herein, or a conservative variant thereof
• an O-RS comprises an amino acid sequence as set forth in the sequence listing, or a conservative variation thereof.
• the O-RS or a portion thereof is encoded by a polynucleotide sequence encoding an amino acid sequence as set forth in the sequence listing or examples herein, or a complementary polynucleotide sequence thereof.
• a test nucleic acid is said to specifically hybridize to a probe nucleic acid when it hybridizes at least V ⁇ as well to the probe as to the perfectly matched complementary target, i.e., with a signal to noise ratio at least l A as high as hybridization of the probe to the target under conditions in which the perfectly matched probe binds to the perfectly matched complementary target with a signal to noise ratio that is at least about 5x-10x as high as that observed for hybridization to any of the unmatched target nucleic acids.
• Stringent hybridization wash conditions in the context of nucleic acid hybridization experiments such as Southern and northern hybridizations are sequence dependent, and are different under different environmental parameters. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993), supra and in Hames and Higgins, 1 and 2. Stringent hybridization and wash conditions can easily be determined empirically for any test nucleic acid. For example, in determining stringent hybridization u a was ⁇ " cond ⁇ f ⁇ bns, '' the Hybridization and wash conditions are gradually increased (e.g., by increasing temperature, decreasing salt concentration, increasing detergent concentration and/or increasing the concentration of organic solvents such as formalin in the hybridization or wash) until a selected set of criteria are met.
• nucleic acids which do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This occurs, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.
• the invention also provides target nucleic acids which hybridize under stringent conditions to a unique coding oligonucleotide which encodes a unique subsequence in a polypeptide selected from the sequences of O-RSs, wherein the unique subsequence is unique as compared to a polypeptide corresponding to any of the control polypeptides (e.g., parental sequences from which synthetases of the invention were derived, e.g., by mutation). Unique sequences are determined as noted above.
• nucleic acid or polypeptide sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (or other algorithms available to persons of skill) or by visual inspection.
• nucleic acids or polypeptides refers to two or more sequences or subsequences that have at least about 60%, about 80%, about 90-95%, about 98%, or about 99% or more nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection.
• sequence comparison algorithm or by visual inspection.
• nucleic acid and virtually any labeled nucleic acid, whether standard or non-standard
• the cell is grown in an appropriate medium.
• the brominated or iodinated amino acid (or other heavy atom-containing amino acid) is provided and incorporated into the specified position in the protein during translation of the nucleic acid with the at least one selector codon, thereby producing the protein.
• a protein produced by this method is also a feature of the invention.
• a heavy atom-containing amino acid e.g., a brominated or iodinated amino acid
• a heavy atom-containing amino acid e.g., a brominated or iodinated amino acid
• the heavy atom amino acid-containing protein is optionally part of a protein crystal.
• Host cells are genetically engineered (e.g., transformed, transduced or transfected) with one or more vectors that express the orthogonal tRNA, the orthogonal tRNA synthetase, and a vector that encodes the protein to be derivatized.
• Each of these components can be on the same vector, or each can be on a separate vector, or two components can be on one vector and the third component on a second vector.
• the vector can be, for example, in the form of a plasmid, a bacterium, a virus, a naked polynucleotide, or a conjugated polynucleotide.
• antibody includes, but is not limited to a polypeptide substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof which specifically bind and recognize an analyte (antigen). Examples include polyclonal, monoclonal, chimeric, and single chain antibodies, and the like. Fragments of immunoglobulins, including Fab fragments and fragments produced by an expression library, including phage display, are also included in the term "antibody” as used herein.
• one general class of embodiments provides methods of determining a protein structure, hi the methods, a protein with a heavy atom-containing amino acid at a specified position is provided by expressing the protein in a translation system that includes an orthogonal tRNA and an orthogonal aminoacyl-tRNA synthetase which preferentially aminoacylates the orthogonal tRNA with the heavy atom-containing amino acid.
• the protein including the heavy atom-containing amino acid is crystallized, thereby creating a heavy atom-containing protein crystal.
• Diffraction data is collected from the heavy atom- containing protein crystal and used to determine the structure, e.g., by MIR, SIR, MAD, SAD, or a combination thereof.
• Electro-competent BL21 (DE3) cells cotransformed with pT4L153TAG and pBK- IodoPheRS were grown in minimal medium containing 1% glycerol and 0.3 mM leucine (GMML medium) with 50 ⁇ g/ml kanamycin, 34 ⁇ g/ml of chloramphenicol, and 1.0 mM - iodo-L-phenylalanine at 37 °C.
• GMML medium 1% glycerol and 0.3 mM leucine

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• 2005
  • 2005-05-24 AU AU2005248392A patent/AU2005248392A1/en not_active Abandoned
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