WO2004104194A1 - Compositions et methodes de recombinaison et d'expression specifiques d'un site - Google Patents

Compositions et methodes de recombinaison et d'expression specifiques d'un site Download PDF

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WO2004104194A1
WO2004104194A1 PCT/US2003/018219 US0318219W WO2004104194A1 WO 2004104194 A1 WO2004104194 A1 WO 2004104194A1 US 0318219 W US0318219 W US 0318219W WO 2004104194 A1 WO2004104194 A1 WO 2004104194A1
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nucleic acid
acid molecule
recombination
methods
sequence
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PCT/US2003/018219
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English (en)
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Aya Miura
Ryohei Satoh
Nobuo Nomura
Naoki Goshima
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Invitrogen Corporation
National Institute Of Advanced Industrial Science And Technology (Aist), Japan Biological Information Research Center (Jbirc)
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Priority to AU2003239211A priority Critical patent/AU2003239211A1/en
Publication of WO2004104194A1 publication Critical patent/WO2004104194A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/64—General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • 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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • 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

  • the present invention is in the fields of molecular biology and protein chemistry.
  • the invention relates to in vitro production of proteins.
  • Methods of the invention may include one or more recombination reactions to create one or more nucleic acid molecules, which may then be used as templates for in vitro transcription and translation.
  • in vitro protein synthesis has among its advantages specifically producing desired proteins without producing undesired proteins that may be required for maintaining cells used for protein production in in vivo or cellular systems.
  • Cell-free systems are very popular because there are standard protocols available for their preparation and because they are commercially available from a number of sources.
  • in vitro protein synthesis has advantages in the production of cytotoxic, unstable and/or insoluble proteins.
  • the over-production of protein beyond a predetermined concentration can be difficult to obtain in vivo, because the expression levels are often regulated by the intracellular concentration of product.
  • the concentration of protein accumulated in the cell generally affects the viability of the cell, so that over-production ofthe desired protein is often difficult to obtain.
  • RNA polynucleotide template that encodes the desired protein.
  • a DNA template comprising a sequence of interest encoding the desired protein is transcribed to produce the RNA polynucleotide that encodes the desired protein.
  • the DNA template therefore minimally includes the DNA sequence of interest to be transcribed as well as a binding site for RNA polymerase (e.g., a promoter) that catalyzes transcription of the mRNA template that is subsequently translated to produce protein.
  • RNA polymerase e.g., a promoter
  • IVTT or protein synthesis using cell-free extracts is becoming an important tool for analysis of proteins.
  • the availability of complete genome sequences for a variety of organisms provides a wealth of information on the molecular structure and organization of a myriad of genes and open reading frames whose functions are not known or are only poorly understood.
  • the utility of IVTT and more generally, protein synthesis in vitro is expected to be even more important in the future for rapid and efficient protein synthesis and functional analysis.
  • cell extracts were developed which allowed the synthesis of protein in vitro from purified mRNA transcripts. To date, several systems have become available for the study of protein synthesis and RNA structure and function.
  • a translation extract must be "programmed" with an mRNA corresponding to the gene and protein under investigation.
  • the mRNA can be produced from DNA, or the mRNA can be added exogenously in purified form.
  • mRNA templates were purified from natural sources or, using more recently developed technologies, prepared synthetically from cloned DNA using bacteriophage RNA polymerases in an in vitro reaction.
  • RNA polymerase which may be prokaryotic (e.g., E. coli RNA polymerase) or eukaryotic (e.g., wheat germ RNA polymerase) in origin, to transcribe exogenous DNA.
  • prokaryotic e.g., E. coli RNA polymerase
  • eukaryotic e.g., wheat germ RNA polymerase
  • Other coupled systems have been developed for the study of viral protein synthesis, but are not generally useful for non-viral templates.
  • E. coli extract for cell-free protein synthesis has been made using a
  • RecD mutant of E. coli (Lesley et al, J. Biol. Chem., 266: 2632-2639 (1991)).
  • An RNase I deficient mutant has been used as a potential source of material for IVTT, but with unsatisfactory results with respect to efficient protein production (Kudlicki et al, Anal. Biochem., 206: 389-393 (1992); and ⁇ llman et al, Methods in Enzymology, 202: 301-336 (1991)).
  • WO 00/55353 discloses two methods for replenishing ATP necessary for translation in an in vitro expression system. According to these methods, PEP (phosphoenolpyruvate) or pyruvate is used to regenerate the ATP energy source.
  • WO 00/55353 The first method disclosed in WO 00/55353 was previously known in the art and involves PEP used in conjunction with pyruvate kinase to regenerate ATP from ADP.
  • PEP used in conjunction with pyruvate kinase to regenerate ATP from ADP.
  • pyruvate is used in conjunction with pyruvate oxidase to regenerate ATP.
  • 0168706 Al describe an improved IVTT system that enhances the production of proteins.
  • the system disclosed provides compositions and methods for stabilizing or maintaining template DNA, stabilizing or maintaining mRNA including mRNA derived from the template(s), conserving energy to be used for synthesis and/or providing sufficient energy regenerating substrates to provide the energy necessary for efficient protein synthesis from the templates.
  • art-known methods still require the construction of suitable input DNA template from which transcription may occur. This is typically accomplished using conventional cloning techniques.
  • a typical method may require restriction digestion of a DNA source and vector, isolation of desired fragments, ligation of the desired fragments with a vector, transformation of a competent host, screening of the transformed hosts to identify a desired clone, growth of a stock of host transformed with the desired clone, and isolation of cloned plasmid DNA from the transformed host.
  • This process may be shortened somewhat using more recently developed cloning techniques such as topoisomerase cloning (see, for example, United States patent no.
  • ORFs open reading frames
  • methods for the production of proteins encoded by these ORFs should be amenable to high throughput applications in order to facilitate the analysis.
  • IVTT open reading frames
  • Art-known methods involving IVTT are limited in utility by the methods used to construct an input DNA template molecule from which transcription can occur.
  • the present invention relates to materials and methods for the construction of nucleic acid molecules (e.g., DNA molecules)and, optionally, the use of nucleic acid molecules produced using materials and methods of the invention in the production of nucleic acid molecules (e.g., DNA and/or RNA molecules) and/or proteins.
  • nucleic acid molecules e.g., DNA molecules
  • nucleic acid molecules e.g., DNA and/or RNA molecules
  • one or more methods of the invention may be practiced in vitro.
  • methods of the invention may be practiced in vitro.
  • the present invention also relates to nucleic acid molecules and/or proteins generated using methods of the invention and to compositions comprising such nucleic acid molecules and/or proteins.
  • the present invention also includes kits comprising components useful in practicing the methods of the invention as well as reaction mixtures used to perform such methods.
  • the present invention provides methods of producing nucleic acid molecules (e.g., DNA and/or RNA molecules) and/or polypeptides. Such methods may include contacting a first nucleic acid molecule, comprising a sequence of interest, which may encode one or more (e.g., two, three, four, five, six, eight, ten, etc.) polypeptides or portion thereof, and comprising one or more (e.g., two, three, four, five, six, eight, ten, etc.) recombination sites, with a second nucleic acid molecule comprising at least one (e.g., two, three, four, five, six, eight, ten, etc.) recombination sites under conditions causing recombination of the first and second nucleic acid molecules to produce a third nucleic acid molecule.
  • a first nucleic acid molecule comprising a sequence of interest, which may encode one or more (e.g., two, three, four, five, six, eight, ten
  • the third nucleic acid molecule may be used as a template in subsequent reactions.
  • a third nucleic acid molecule generated using methods of the invention may be suitable for logarithmic amplification (e.g., by PCR) and/or may be suitable for use as a template in an in vitro transcription reaction.
  • a third nucleic acid molecule may be amplified (e.g., by PCR) and the amplification product may be used as a template for an in vitro transcription reaction.
  • a second nucleic acid molecule may comprise one or more (e.g., two, three, four, five, six, eight, ten, etc.) transcriptional regulatory sequences.
  • a third nucleic acid molecule produced using methods of the invention may comprise a transcriptional regulatory sequence and a sequence of interest, which may be operably linked.
  • methods of the invention may be used to generate a third nucleic acid molecule comprising a transcriptional regulatory sequence operably linked to a sequence of interest.
  • such a third nucleic acid molecule may be transcribed into RNA.
  • RNA corresponding to a third nucleic acid molecule may be used, for example, as RNA (e.g., antisense RNA, siRNA, etc.) or may be translated into one or more polypeptides.
  • RNA e.g., antisense RNA, siRNA, etc.
  • all or a portion of the sequence of interest may be transcribed into RNA.
  • the RNA thus produced may be translated into one or more polypeptides.
  • transcription and/or translation may be done in vitro.
  • a third nucleic acid molecule generated according to methods of the invention is not introduced into a cell.
  • a third nucleic acid molecule generated by methods of the invention may be amplified and the amplification product used in an in vitro transcription reaction to produce RNA and the RNA translated in vitro into one or more polypeptides all without introduction of any of the molecules involved (e.g., first nucleic acid molecule, second nucleic acid molecule, third nucleic acid molecule, amplification product, corresponding RNA) being introduced into a cell.
  • the second nucleic acid molecule may be an expression vector.
  • a second nucleic acid molecule may be a closed circle, which may be supercoiled, or may be linear.
  • a closed circular nucleic acid molecule may be converted to a linear molecule, for example, by digestion with one or more restriction enzymes.
  • Linear second nucleic acid molecules may be made by other methods known to those skilled in the art, for example, by PCR.
  • the second nucleic acid molecule may be an expression vector and may comprise at least two recombination sites. The second nucleic acid molecule may be digested with one or more restriction enzyme prior to recombination with a first nucleic acid molecule.
  • restriction enzyme recognizing a site in the second nucleic acid molecule may be used in these embodiments.
  • restriction enzymes suitable for use in methods of the invention include, but are not limited to, BgHl and Pstl.
  • the second nucleic acid molecule may be a vector having two recombination sites and at least one site recognized by a restriction enzyme is located between the two recombination sites. Digestion of such a second nucleic acid molecule results in a linear nucleic acid molecule in which the recombination sites are "tethered" (i.e., are in the same nucleic acid molecule).
  • a second nucleic acid molecule When such a second nucleic acid molecule is reacted in a recombination reaction with a first nucleic acid molecule comprising two recombination sites, the tethered sites are re-circularized to produce a third nucleic acid molecule that can be amplified using primers that hybridize to the second nucleic acid molecule such that no amplification product can be produced in the absence of a recombination reaction.
  • transcriptional regulatory sequences in second nucleic acid molecules for use in the present invention may comprise a promoter.
  • Any promoter known to those skilled in the art may be used, for example, the promoter may be a phage promoter.
  • Promoters suitable for use in methods of the invention include, but are not limited to, phage promoters, viral promoters, and other promoters known to those skilled in the art. Examples include, but are not limited to, the T7 promoter and the SP6 promoter.
  • Other transcriptional regulatory sequences suitable for use in the present invention include, but are not limited to, enhancers, repressors, operators, suppressors, and the like.
  • methods of the invention may comprise amplifying all or a portion ofthe third nucleic acid molecule.
  • the present invention provides methods of making one or more desired nucleic acid molecules. Such methods may include contacting a first nucleic acid molecule comprising a sequence of interest, which may encode one or more polypeptides or may not encode a polypeptide, and comprising one or more recombination sites, with a second nucleic acid molecule comprising one or more recombination sites and, optionally, one or more transcriptional regulatory sequence, under conditions causing recombination of the first and second nucleic acid molecules to produce a desired nucleic acid molecule.
  • the sequence of interest and transcriptional regulatory sequence when present
  • a method of this type may entail amplifying all or a portion of the desired nucleic acid molecule. In one such aspect, the desired nucleic acid molecule is not introduced into a cell before being amplified.
  • the second nucleic acid molecule may be an expression vector and may be circular or linear.
  • the second nucleic acid molecule is an expression vector and is digested with one or more restriction enzymes prior to recombination. Any restriction enzyme recognizing a site in the second nucleic acid molecule may be used in these embodiments.
  • restriction enzymes suitable for use in methods of the invention include, but are not limited to, Bgl ⁇ l and Pstl.
  • a second nucleic acid molecule may be a vector having two or more recombination sites and at least one site recognized by a restriction enzyme is located between at least two recombination sites.
  • transcriptional regulatory sequences in second nucleic acid molecules for use in the present invention may comprise a promoter.
  • Promoters suitable for use in methods of the invention include, but are not limited to, phage promoters, viral promoters, and other promoters known to those skilled in the art. Examples include, but are not limited to, the T7 promoter and the SP6 promoter.
  • Other transcriptional regulatory sequences suitable for use in the present invention include, but are not limited to, enhancers, repressors, operators, suppressors, and the like.
  • the present invention provides methods of amplifying one or more nucleic acid molecules. Such methods may include recombining one or more first nucleic acid molecules and one or more second nucleic acid molecules to form one or more third nucleic acid molecules, which may be amplified, for example, by PCR. Methods of this type may entail mixing one or more third nucleic acid molecules with one or more primers and one or more polypeptides having polymerase activity to form a mixture and incubating the mixture under conditions sufficient to amplify the one or more third nucleic acid molecules.
  • one or more third nucleic acid molecules may be double stranded nucleic acid molecules and such amplification methods may include contacting a first strand of the third nucleic acid molecule with a first primer molecule which is complementary to a portion of the first strand; and contacting a second strand of the third nucleic acid molecule with a second primer molecule which is complementary to a portion of said second strand in the presence of one or more polypeptides having polymerase activity to form a mixture; and incubating the mixture under conditions sufficient to form a third strand complementary to all or a portion of the first strand and a fourth strand complementary to all or a portion of the second strand.
  • Such methods may also include denaturing the first and third and the second and fourth strands and repeating the contacting and incubating described above.
  • such conditions according to the invention may include incubating the mixture in the presence of one or more nucleotides, one or more buffers or buffering salts, one or more primers, one or more cofactors, and/or one or more polypeptides having a nucleotide polymerase activity.
  • compositions for carrying out methods of the invention also relates to compositions for carrying out methods of the invention and to compositions made while carrying out methods of the invention.
  • Such compositions may comprise any one or a combination of the elements used in methods of the invention (e.g. one or more recombination proteins, one or more restriction enzymes, one or more promoter sequences, etc.) and/or they may also comprise one or more substrates used during transcription and/or translation reactions (e.g. one or more nucleotides, one or more amino acids, one or more polymerases, one or more enzymes, one or more cofactors, one or more buffers and one or more buffering salts).
  • the elements used in methods of the invention e.g. one or more recombination proteins, one or more restriction enzymes, one or more promoter sequences, etc.
  • substrates used during transcription and/or translation reactions e.g. one or more nucleotides, one or more amino acids, one or more polymerases, one or more enzymes, one
  • compositions may comprise one or more products of the transcription and/or translation reactions such as RNA, peptides, proteins, etc.
  • the compositions ofthe invention comprise at least one component selected from the group consisting of one or more recombination proteins, one or more restriction enzymes, one or more polymerase enzymes, and one or more cell extracts.
  • kits for practicing the methods of the invention may comprise one or more containers.
  • Such containers may contain a variety of components, for example, one or more recombination proteins, one or more restriction enzymes, and/or one or more cell extracts for in vitro translation.
  • kits may comprise one or more containers containing one or more recombination proteins, one or more containers containing one or more restriction enzymes, and one or more containers containing one or more cell extracts.
  • Kits of the invention may comprises one or more additional components such as one or more containers containing one or more components selected from the group consisting of one or more polymerases, one or more buffers, one or more primers, one or more vectors, one or more nucleic acid molecules comprising one or more promoter sequences, and one or more nucleotides.
  • Kits for in vitro synthesis of nucleic acid molecules and/or polypeptides are also provided by the present invention.
  • kits may contain any number or combination of reagents or components for carrying out the invention.
  • Kits of the invention preferably comprise one or more elements selected from the group consisting of one or more nucleotides or derivatives thereof; one or more amino acids or derivatives thereof; one or more transfer RNA molecules, which may be charged with an amino acid; one or more polymerases; one or more cofactors; one or more buffers or buffer salts; one or more energy sources; one or more cell extracts; one or more nucleic acid templates; one or more reagents to determine the efficiency of the kit or assay for production of the products such as nucleic acid and protein products; and directions or protocols for carrying out the methods of the invention or to use of the kits of the invention and/or its components.
  • kits of the invention may comprise one or more of the above components in any number of separate containers, tubes, vials and the like or such components may be combined
  • the present invention relates to methods (e.g., business methods) for providing a product suitable for the practice of one or more of the previously-described methods of the invention (e.g., methods of making polypeptides, methods of making nucleic acid molecules, etc.) to a customer.
  • Such methods for providing a product may comprise receiving an order for a product from a customer; and providing the customer with the product.
  • a product may be a kit for practicing one or more of the methods of the invention.
  • a kit may comprise one or more containers comprising one or more recombination proteins and one or more containers comprising one or more cell extracts.
  • kits may comprise written instructions.
  • An order received in the practice of the present method may be received by any mode of communication, for example, by telephone, via the internet, etc.
  • the present invention encompasses methods for providing information to a customer as to availability of a product suitable for the practice of a method ofthe invention (e.g., method of making a polypeptide, method of making a nucleic acid molecule, etc.).
  • a method of providing information may comprise transmitting product description data to an output source; transmitting to the output source instructions to publish the product description in media accessible to potential interested parties; and detecting publication of the data in the media, thereby providing information as to availability of the product to parties having potential interest in the availability of the product.
  • a product may be a kit.
  • kit may comprise one or more containers comprising one or more recombination proteins and one or more containers comprising one or more cell extracts.
  • kit may further comprise written instructions.
  • product description data may be published in a print medium and/or may be published on the internet.
  • Fig. 1 is a schematic representation of a recombinational cloning reaction.
  • Fig. 2 is a schematic representation of the recombination region of a
  • Fig. 3 is a schematic representation of the arrangement of sequences that may be included in a T7 promoter-containing plasmid ofthe invention.
  • Fig. 4 is a schematic representation of a system for providing a product to a party.
  • Fig. 5 provides a schematic representation of a system for advising a party as to the availability of a product.
  • Figs. 6 A and 6B provide a comparison of the present invetion to previously known methods.
  • Fig. 6A is a timeline comparing methods of the present invention to previously available in vivo methods.
  • Fig. 6A is a timeline comparing methods of the present invention to previously available in vivo methods.
  • FIG. 6B shows a schematic representation of the reactions performed in on embodiment of the present invention.
  • Fig. 7 is a schematic representation of a recombination reaction that may be performed in the practice ofthe present invention.
  • Fig. 8 is a schematic representation of a high throughput in vitro synthesis reaction in accordance with the present invention. DETAILED DESCRIPTION OF THE INVENTION
  • Host refers to any prokaryotic or eukaryotic (e.g., mammalian, insect, yeast, plant, avian, animal, etc.) organism that is a recipient of a replicable expression vector, cloning vector or any nucleic acid molecule.
  • the nucleic acid molecule may contain, but is not limited to, a sequence of interest, a transcriptional regulatory sequence (such as a promoter, enhancer, repressor, and the like) and/or an origin of replication.
  • the terms "host,” “host cell,” “recombinant host” and “recombinant host cell” may be used interchangeably. For examples of such hosts, see Sambrook, et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.
  • transcriptional regulatory sequence refers to any functional stretch of nucleotides contained on a nucleic acid molecule, in any configuration or geometry, that act to regulate the synthesis of an RNA molecule from a DNA template molecule (i.e., transcription).
  • transcriptional regulatory sequences include, but are not limited to, promoters, enhancers, repressors, operators (e.g., the tet operator), suppressors and the like.
  • a promoter is an example of a transcriptional regulatory sequence, and more specifically is a nucleic acid generally described as the 5'-region of a gene located proximal to the start codon or nucleic acid that encodes untranslated RNA. The transcription of an adjacent nucleic acid segment is initiated at or near the promoter. A repressible promoter's rate of transcription decreases in response to a repressing agent. An inducible promoter's rate of transcription increases in response to an inducing agent. A constitutive promoter's rate of transcription is not specifically regulated, although it can vary under the influence of general metabolic conditions.
  • recognition sequence refers to a particular nucleotide sequence that is recognized by and bound by a protein, chemical compound, DNA, or RNA molecule (e.g., restriction endonuclease, a modification methylase, topoisomerases, a recombination protein or a recombinase).
  • a recognition sequence may refer to a recombination site.
  • the recognition sequence for Cre recombinase is loxP, which is a 34 base pair sequence comprising two 13 base pair inverted repeats (serving as the recombinase binding sites) flanking an 8 base pair core sequence (see Fig.
  • ⁇ ttB is an approximately 25 base pair sequence containing two 9 base pair core-type Int binding sites and a 7 base pair overlap region.
  • ⁇ ttP is an approximately 240 base pair sequence containing core-type Int binding sites and arm-type Int binding sites as well as sites for auxiliary proteins such as integration host factor (LHF), Fis and excisionase (Xis) (see Landy, Current Opinion in Biotechnology 3:699-707 (1993)).
  • LHF integration host factor
  • Xis excisionase
  • Such sites may also be engineered according to the present invention to enhance production of products in the methods of the invention.
  • engineered sites lack the PI or HI domains to make the recombination reactions irreversible (e.g., ⁇ ttR or ⁇ ttP)
  • such sites may be designated ⁇ ttR' or ⁇ ttP' to show that the domains of these sites have been modified in some way.
  • Recombination proteins includes excisive or integrative proteins, enzymes, co-factors or associated proteins that are involved in recombination reactions involving one or more recombination sites (e.g., two, three, four, five, seven, ten, twelve, fifteen, twenty, thirty, fifty, etc.), which may be wild-type proteins (see Landy, Current Opinion in Biotechnology 3:699-707 (1993)), or mutants, derivatives (e.g., fusion proteins containing the recombination protein sequences or fragments thereof), fragments, and variants thereof.
  • recombination proteins includes excisive or integrative proteins, enzymes, co-factors or associated proteins that are involved in recombination reactions involving one or more recombination sites (e.g., two, three, four, five, seven, ten, twelve, fifteen, twenty, thirty, fifty, etc.), which may be wild-type proteins (see Landy, Current Opinion in Biotechnology 3:699-707 (1993)), or mutant
  • recombination proteins include Cre, Int, IHF, Xis, Flp, Fis, Hin, Gin, ⁇ C31 , Cin, Tn3 resolvase, TndX, XerC, XerD, TnpX, Hjc, SpCCEl, and ParA.
  • Recombinases As used herein, the term “recombinases” is used to refer to the protein that catalyzes strand cleavage and re-ligation in a recombination reaction.
  • Site-Specific Recombinase refers to a type of recombinase that typically has at least the following four activities (or combinations thereof): (1) recognition of specific nucleic acid sequences; (2) cleavage of said sequence or sequences; (3) topoisomerase activity involved in strand exchange; and (4) ligase activity to reseal the cleaved strands of nucleic acid (see Sauer, B., Current Opinions in Biotechnology 5:521-527 (1994)).
  • Conservative site-specific recombination is distinguished from homologous recombination and transposition by a high degree of sequence specificity for both partners.
  • the strand exchange mechanism involves the cleavage and rejoining of specific nucleic acid sequences in the absence of DNA synthesis (Landy, A. (1989) Ann. Rev. Biochem. 55:913-949).
  • Site-specific recombinases are proteins that are present in many organisms (e.g., viruses and bacteria). These recombinases (along with associated proteins in some cases) recognize specific sequences of bases in a nucleic acid molecule and exchange the nucleic acid segments flanking those sequences.
  • the recombinases and associated proteins are collectively referred to as "recombination proteins” (see, e.g., Landy, A., Current Opinion in Biotechnology 3:699-707 (1993)).
  • Recombination site refers to a recognition sequence on a nucleic acid molecule that participates in an integration/recombination reaction by recombination proteins. Recombination sites are discrete sections or segments of nucleic acid on the participating nucleic acid molecules that are recognized and bound by a site- specific recombination protein during the initial stages of integration or recombination.
  • recombination sites include the ⁇ ttB, ⁇ ttP, ⁇ ttL, and ⁇ ttR sequences described in co-pending United States patent applications 09/517,466, filed March 2, 2000, and 09/732,914, filed December 11, 2000 (published as US 2002/0007051 Al)— all of which are specifically incorporated herein by reference — and mutants, fragments, variants and derivatives thereof, which are recognized by the recombination protein ⁇ Int and/or by the auxiliary proteins integration host factor (IHF), FIS and excisionase (Xis) (see Landy, Curr. Opin. Biotech. 3:699-707 (1993)).
  • IHF auxiliary proteins integration host factor
  • FIS FIS
  • Xis excisionase
  • Recombination sites may be added to molecules by any number of known methods. For example, recombination sites can be added to nucleic acid molecules by blunt end ligation, PCR performed with fully or partially random primers, or inserting the nucleic acid molecules into an vector using a restriction site flanked by recombination sites.
  • recombinational cloning refers to a method, such as that described in U.S. Patent Nos. 5,888,732; 6,143,557; 6,171,861; 6,270,969; and 6,277,608 (the contents of which are fully incorporated herein by reference), whereby segments of nucleic acid molecules or populations of such molecules are exchanged, inserted, replaced, substituted or modified, in vitro or in vivo.
  • such cloning method is an in vitro method.
  • Figure 1 provides a schematic representation of a recombinational cloning reaction between two closed circular nucleic acid molecules.
  • Recombination sites are represented as squares or circles.
  • Recombination sites capable of recombining with each other are shown as the same shape (i.e., squares recombine with squares and circles recombine with circles).
  • a recombination reaction occurs between sites capable of recombining with each other producing a nucleic acid molecule containing both starting molecules (a cointegrate molecule).
  • Reaction ofthe second pair of recombination sites resolves the cointegrate into two new nucleic acid molecules.
  • a first nucleic acid molecule comprising segments A and B is reacted with a second nucleic acid molecule comprising segments C and D to produce two new nucleic acid molecules, a third nucleic acid molecule comprising segment s A and D, and a fourth nucleic acid molecule comprising segments B and C.
  • Cloning systems that utilize recombination at defined recombination sites have been previously described in U.S. patent no. 5,888,732, U.S. patent no. 6,143,557, U.S. patent no. 6,171,861, U.S. patent no. 6,270,969, and U.S. patent no. 6,277,608, and in pending United States application no.
  • the GATEWAYTM Cloning System described in these patents and applications utilizes vectors that contain at least one recombination site to clone desired nucleic acid molecules in vivo or in vitro.
  • the system utilizes vectors that contain at least two different site-specific recombination sites that may be based on the bacteriophage lambda system (e.g., attl and ⁇ tt2) that are mutated from the wild-type ( ⁇ ttO) sites.
  • Each mutated site has a unique specificity for its cognate partner att site (i.e., its binding partner recombination site) of the same type (for example ⁇ ttBl with ⁇ ttPl, or ⁇ ttLl with ⁇ ttRl) and will not cross-react with recombination sites of the other mutant type or with the wild-type ⁇ ttO site.
  • ⁇ ttBl with ⁇ ttPl, or ⁇ ttLl with ⁇ ttRl binding partner recombination site
  • Different site specificities allow directional cloning or linkage of desired molecules thus providing desired orientation of the cloned molecules.
  • Nucleic acid fragments flanked by recombination sites are cloned and subcloned using the GATEWAYTM system by replacing a selectable marker (for example, an antibiotic resistance gene) flanked by att sites on the recipient plasmid molecule, sometimes termed the Destination Vector. Desired clones are then selected by transformation of a host strain and positive selection for a selectable marker on the recipient molecule. Similar strategies for negative selection (e.g., use of toxic genes such as ccdB) can be used in other organisms such as thymidine kinase (TK) in mammals and insects.
  • TK thymidine kinase
  • Mutating specific residues in the core region ofthe att site can generate a large number of different att sites.
  • each additional mutation potentially creates a novel att site with unique specificity that will recombine only with its cognate partner att site bearing the same mutation and will not cross-react with any other mutant or wild-type att site.
  • Novel mutated att sites e.g., ⁇ ttB 1-10, ⁇ ttP 1-10, ⁇ ttR 1-10 and ⁇ ttL 1-10) are described in previous patent application serial number 09/517,466, filed March 2, 2000, which is specifically incorporated herein by reference.
  • recombination sites having unique specificity i.e., a first site will recombine with its corresponding site and will not recombine or not substantially recombine with a second site having a different specificity
  • suitable recombination sites include, but are not limited to, loxP sites; loxP site mutants, variants or derivatives such as loxP511 (see U.S. Patent No.
  • Repression Cassette refers to a nucleic acid segment that contains a repressor or a selectable marker present in the subcloning vector.
  • Vector refers to a nucleic acid molecule (preferably DNA) that provides a useful biological or biochemical property to an insert. Examples include plasmids, phages, autonomously replicating sequences (ARS), centromeres, and other sequences that are able to replicate or be replicated in vitro or in a host cell, or to convey a desired nucleic acid segment to a desired location within a host cell.
  • a vector can have one or more recognition sites (e.g., two, three, four, five, seven, ten, etc.
  • Vectors can further provide primer sites (e.g., for PCR), transcriptional and/or translational initiation and/or regulation sites, recombinational signals, replicons, selectable markers, etc.
  • cloning vector can further contain one or more selectable markers (e.g., two, three, four, five, seven, ten, etc.) suitable for use in the identification of cells transformed with the cloning vector.
  • Subcloning vector refers to a cloning vector comprising a circular or linear nucleic acid molecule that includes, preferably, an appropriate replicon.
  • the subcloning vector can also contain functional and/or regulatory elements that are desired to be incorporated into the final product to act upon or with the cloned nucleic acid insert.
  • the subcloning vector can also contain a selectable marker (preferably DNA).
  • Primer refers to a single stranded or double stranded oligonucleotide that is extended by covalent bonding of nucleotide monomers during amplification or polymerization of a nucleic acid molecule (e.g., a DNA molecule).
  • the primer may be a sequencing primer (for example, a universal sequencing primer).
  • the primer may comprise a recombination site or portion thereof.
  • templates refers to a double stranded or single stranded nucleic acid molecule that serves to direct synthesis of another nucleic acid molecule.
  • Examples of synthesis of another nucleic acid molecule from a template include, but are not limited to, amplification, transcription, and sequencing.
  • denaturation of its strands to form a first and a second strand is preferably performed before these molecules may be amplified, synthesized or sequenced, or the double stranded molecule may be used directly as a template.
  • a primer complementary to at least a portion of the template hybridizes under appropriate conditions and one or more polypeptides having polymerase activity (e.g., two, three, four, five, or seven DNA polymerases and/or reverse transcriptases) may then synthesize a molecule complementary to all or a portion of the template.
  • one or more transcriptional regulatory sequences e.g., two, three, four, five, seven or more promoters
  • the newly synthesized molecule may be of equal or shorter length compared to the original template.
  • Mismatch incorporation or strand slippage during the synthesis or extension of the newly synthesized molecule may result in one or a number of mismatched base pairs.
  • the synthesized molecule need not be exactly complementary to the template.
  • a population of nucleic acid templates may be used during synthesis or amplification to produce a population of nucleic acid molecules typically representative ofthe original template population.
  • incorporasing means becoming a part of a nucleic acid (e.g., DNA) molecule or primer.
  • Library refers to a collection of nucleic acid molecules (circular or linear).
  • a library may comprise a plurality of nucleic acid molecules (e.g., two, three, four, five, seven, ten, twelve, fifteen, twenty, thirty, fifty, one hundred, two hundred, five hundred one thousand, five thousand, or more), that may or may not be from a common source organism, organ, tissue, or cell.
  • a library is representative of all or a portion or a significant portion of the nucleic acid content of an organism (a "genomic” library), or a set of nucleic acid molecules representative of all or a portion or a significant portion of the expressed nucleic acid molecules (a cDNA library or segments derived therefrom) in a cell, tissue, organ or organism.
  • a library may also comprise nucleic acid molecules having random sequences made by de novo synthesis, mutagenesis of one or more nucleic acid molecules, and the like.
  • Such libraries may or may not be contained in one or more vectors (e.g., two, three, four, five, seven, ten, twelve, fifteen, twenty, thirty, fifty, etc.).
  • Amplification refers to any in vitro method for increasing the number of copies of a nucleic acid molecule with the use of one or more polypeptides having polymerase activity (e.g., one, two, three, four or more nucleic acid polymerases or reverse transcriptases). Nucleic acid amplification results in the incorporation of nucleotides into a DNA and/or RNA molecule or primer thereby forming a new nucleic acid molecule complementary to a template. The formed nucleic acid molecule and its template can be used as templates to synthesize additional nucleic acid molecules. As used herein, one amplification reaction may consist of many rounds of nucleic acid replication. DNA amplification reactions include, for example, polymerase chain reaction (PCR). One PCR reaction may consist of 5 to 100 cycles of denaturation and synthesis of a DNA molecule.
  • PCR polymerase chain reaction
  • nucleotide refers to a base- sugar-phosphate combination. Nucleotides are monomeric units of a nucleic acid molecule (DNA and RNA).
  • the term nucleotide includes ribonucleoside triphosphates ATP, UTP, CTG, GTP and deoxyribonucleoside triphosphates such as dATP, dCTP, dITP, dUTP, dGTP, dTTP, or derivatives thereof. Such derivatives include, for example, [ ⁇ -S]dATP, 7-deaza-dGTP and 7-deaza- dATP.
  • nucleotide as used herein also refers to dideoxyribonucleoside triphosphates (ddNTPs) and their derivatives. Illustrated examples of dideoxyribonucleoside triphosphates include, but are not limited to, ddATP, ddCTP, ddGTP, ddlTP, and ddTTP. According to the present invention, a "nucleotide" may be unlabeled or detectably labeled by well known techniques. Detectable labels include, for example, radioactive isotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels and enzyme labels.
  • nucleic acid molecule refers to a sequence of contiguous nucleotides (riboNTPs, dNTPs, ddNTPs, or combinations thereof) of any length.
  • a nucleic acid molecule may encode a full-length polypeptide or a fragment of any length thereof, or may be non-coding.
  • nucleic acid molecule and polynucleotide may be used interchangeably and include both RNA and DNA.
  • Oligonucleotide refers to a synthetic or natural molecule comprising a covalently linked sequence of nucleotides that are joined by a phosphodiester bond between the 3' position of the pentose of one nucleotide and the 5' position of the pentose of the adjacent nucleotide.
  • Protein refers to a sequence of contiguous amino acids of any length.
  • peptide oligopeptide
  • polypeptide may be used interchangeably herein with the term “protein.”
  • Hybridization As used herein, the terms “hybridization” and
  • hybridizing refers to base pairing of two complementary single-stranded nucleic acid molecules (RNA and/or DNA) to give a double stranded molecule.
  • RNA and/or DNA complementary single-stranded nucleic acid molecules
  • two nucleic acid molecules may hybridize, although the base pairing is not completely complementary. Accordingly, mismatched bases do not prevent hybridization of two nucleic acid molecules provided that appropriate conditions, well known in the art, are used.
  • hybridization is said to be under "stringent conditions.”
  • stringent conditions as the phrase is used herein, is meant overnight incubation at 42°C in a solution comprising: 50% formamide, 5x SSC (750 mM NaCl, 75m M trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 ⁇ g/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1 x SSC at about 65°C.
  • Extract refers to a cell lysate or exudate.
  • the cell can be any cell that can be grown for preparing an extract. Both prokaryotic cells and eukaryotic cells can be used to prepare extracts for protein and/or nucleic acid synthesis according to the invention.
  • An extract may be prepared by lysing cells using techniques known in the art. Preferably, an extract is processed to remove cellular debris. Centrifugation is a common method for removing such solid material. Filtration, chromatography, or any other separation or purification procedures may be used to produce a desired extract.
  • the extract preferably includes all necessary components for practicing the methods of the invention.
  • An extract can be concentrated using one or more of the many tools of the art. One or more components (e.g., enzymes, energy sources, and the like) present in the extract can originate in the extracted cell or can be added during the production of the extract.
  • Extracts may also include a mixture of components crafted to imitate a cell lysate or exudate with respect to the components necessary or desired for protein and/or nucleic acid synthesis.
  • An extract thus can be a mixture of components to imitate or improve upon a cell lysate or exudate in protein synthesis reactions and/or to provide components used for synthesis from a nucleic acid template.
  • such mixtures can be produced by obtaining a partial extract or fraction thereof and/or by mixing any number of individual components.
  • Polymerase refers to an enzyme that catalyses synthesis of nucleic acids using a preexisting nucleic acid template.
  • a polymerase may synthesize an RNA and/or a DNA molecule.
  • Enzymes having nucleic acid polymerase activity suitable for use in the present methods include polymerases that are active in the chosen system with the chosen template to synthesize protein.
  • the extract of a cell will usually contain a suitable polymerase, such as RNA polymerase II, SP6 RNA polymerase, T3 RNA polymerase, T7 RNA polymerase, RNA polymerase III and/or phage-derived RNA polymerases.
  • RNA polymerases are available commercially and/or are known in the art and can be readily accessed by the skilled artisan by searching one or more of the public or private databases. Suitable polymerase can also be supplemented in the system. When RNA is to be synthesized from a DNA template, a polymerase active on the DNA molecule of interest should be used. RNA polymerases and transcription factors useful in the invention are well known in the art and will be readily recognized by those skilled in the art.
  • in vivo means within a living cell or organism.
  • in vitro means outside a living cell or organism (e.g., in a cell extract, in a test tube or other container, etc.).
  • the present invention relates to materials and methods for the in vitro construction of template nucleic acid molecules and their use in in vitro synthesis of proteins.
  • the invention includes synthesis systems, methods and kits embodying one or more of the features of the present invention.
  • the synthesis system of the present invention includes the necessary components to synthesize proteins from template nucleic acid molecules.
  • the in vitro synthesis system of the invention provides efficient synthesis outside the confines of a cell.
  • The. kits of the present invention allow or facilitate the practice of the methods of the invention (e.g., methods of making template nucleic acid molecules, methods of in vitro transcription translation, methods of making polypeptides, etc).
  • methods of the invention may comprise making a nucleic acid molecule (e.g., a DNA molecule) in a process that may involve a site- specific recombination reaction.
  • Nucleic acid molecules made by methods of the invention may be used in further reactions.
  • nucleic acid molecules of the invention may be used in in vitro transcription reactions, in vitro translation reactions, coupled in vitro transcription/translation reactions, amplification reactions (e.g., PCR), and/or combinations of these reactions.
  • a nucleic acid molecule produced by the methods of the invention may be amplified and used in a coupled transcription/translation reaction.
  • Recombination sites for use in the invention may be any nucleic acid that can serve as a substrate in a recombination reaction. Such recombination sites may be wild-type or naturally occurring recombination sites, or modified, variant, derivative, or mutant recombination sites.
  • recombination sites for use in the invention include, but are not limited to, phage-lambda recombination sites (such as ⁇ ttP, ⁇ ttB, ⁇ ttL, and ⁇ ttR and mutants or derivatives thereof) and recombination sites from other bacteriophages such as phi80, P22, P2, 186, P4 and PI (including lox sites such as loxP and loxP511).
  • phage-lambda recombination sites such as ⁇ ttP, ⁇ ttB, ⁇ ttL, and ⁇ ttR and mutants or derivatives thereof
  • recombination sites from other bacteriophages such as phi80, P22, P2, 186, P4 and PI (including lox sites such as loxP and loxP511).
  • Preferred recombination proteins and mutant, modified, variant, or derivative recombination sites for use in the invention include those described in U.S. Patent Nos. 5,888,732, 6,143,557, 6,171,861, 6,270,969, and 6,277,608 and in U.S. application no. 09/438,358, filed November 12, 1999, which are specifically incorporated herein by reference.
  • Mutated att sites are described in United States application numbers 09/517,466, filed March 2, 2000, and 09/732,914, filed December 11, 2000 (published as US 2002/0007051-Al) the disclosures of which are specifically incorporated herein by reference in their entirety.
  • Other suitable recombination sites and proteins are those associated with the GATEWAYTM Cloning Technology systems available from Invitrogen Corporation, Carlsbad, CA, and are described in the associated product literature, the entire disclosures of all of which are specifically incorporated herein by reference in their entireties.
  • Recombination sites that may be used in the present invention include att sites.
  • the 15 bp core region of the wildtype att site (GCTTTTTTAT ACTAA (SEQ ID NO: )), which is identical in all wildtype att sites, may be mutated in one or more positions.
  • Engineered att sites that specifically recombine with other engineered att sites can be constructed by altering nucleotides in and near the 7 base pair overlap region, bases 6-12, of the core region.
  • recombination sites suitable for use in the methods, molecules, compositions, and vectors of the invention include, but are not limited to, those with insertions, deletions or substitutions of one, two, three, four, or more nucleotide bases within the 15 base pair core region (see U.S. Patent Nos. 5,888,732 and 6,277,608, which describe the core region in further detail, and the disclosures of which are incorporated herein by reference in their entireties).
  • Recombination sites suitable for use in the methods, compositions, and vectors of the invention also include those with insertions, deletions or substitutions of one, two, three, four, or more nucleotide bases within the 15 base pair core region that are at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, or at least 95% identical to this 15 base pair core region.
  • nucleic acid molecule is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, a given recombination site nucleotide sequence or portion thereof can be determined conventionally using known computer programs such as DNAsis software (Hitachi Software, San Bruno, California) for initial sequence alignment followed by ESEE version 3.0 DNA/protein sequence software (cabot@trog.mbb.sfu.ca) for multiple sequence alignments.
  • DNAsis software Haitachi Software, San Bruno, California
  • ESEE version 3.0 DNA/protein sequence software cabot@trog.mbb.sfu.ca
  • such determinations may be accomplished using the BESTFIT program (Wisconsin Sequence Analysis Package, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wl 53711), which employs a local homology algorithm (Smith and Waterman, Advances in Applied Mathematics 2: 482-489 (1981)) to find the best segment of homology between two sequences.
  • BESTFIT Garnier-Fidelity
  • nucleic acid molecules suitable for use with the invention also include those comprising insertions, deletions or substitutions of one, two, three, four, or more nucleotides within the seven base pair overlap region (TTTATAC, bases 6-12 in the core region).
  • the overlap region is defined by the cut sites for the integrase protein and is the region where strand exchange takes place.
  • mutants, fragments, variants and derivatives include, but are not limited to, nucleic acid molecules in which (1) the thymine at position 1 of the seven bp overlap region has been deleted or substituted with a guanine, cytosine, or adenine; (2) the thymine at position 2 of the seven bp overlap region has been deleted or substituted with a guanine, cytosine, or adenine; (3) the thymine at position 3 of the seven bp overlap region has been deleted or substituted with a guanine, cytosine, or adenine; (4) the adenine at position 4 of the seven bp overlap region has been deleted or substituted with a guanine, cytosine, or thymine; (5) the thymine at position 5 of the seven bp overlap region has been deleted or substituted with a guanine, cytosine, or adenine; (6) the adenine at position 6 of the seven
  • nucleic acid molecules and methods of the invention include those comprising or employing one, two, three, four, five, six, eight, ten, or more recombination sites which affect recombination specificity, particularly one or more (e.g., one, two, three, four, five, six, eight, ten, twenty, thirty, forty, fifty, etc.) different recombination sites that may correspond substantially to the seven base pair overlap within the 15 base pair core region, having one or more mutations that affect recombination specificity.
  • Particularly preferred such molecules may comprise a consensus sequence such as NNNATAC wherein "N" refers to any nucleotide (i.e., may be A, G, T/U or C, or an analogue or derivative thereof).
  • N refers to any nucleotide (i.e., may be A, G, T/U or C, or an analogue or derivative thereof).
  • each att site ( ⁇ ttB, ⁇ ttP, ⁇ ttL and ⁇ ttR) can be divided into functional units consisting of integrase binding sites, integrase cleavage sites and sequences that determine specificity. Specificity determinants are defined by the first three positions following the integrase top strand cleavage site. These three positions are shown with underlining in the following reference sequence: CAACTTTTTTATAC AAAGTTG (SEQ LD NO: ). Modification of these three positions (64 possible combinations) can be used to generate att sites that recombine with high specificity with other att sites having the same sequence for the first three nucleotides ofthe seven base pair overlap region. The possible combinations of first three nucleotides of the overlap region are shown in Table 1.
  • the invention further includes nucleic acid molecules comprising one or more (e.g., one, two, three, four, five, six, eight, ten, twenty, thirty, forty, fifty, etc.) nucleotides sequences set out in Table 2.
  • nucleic acid molecules comprising the nucleotide sequence GAAATAC, GATATAC, ACAATAC, or TGCATAC.
  • mutated att sites that may be used in the practice of the present invention include ⁇ ttBl (AGCCTGCTTT TTTGTACAAA CTTGT (SEQ LD NO: )), ⁇ ttPl (TACAGGTCAC TAATACCATC TAAGTAGTTG ATTCATAGTG ACTGGATATG TTGTGTTTTA CAGTATTATG
  • TAGTCTGTTT TTTATGCAAA ATCTAATTTA ATATATTGAT ATTTATATCA TTTTACGTTT CTCGTTCAGC TTTTTTGTAC AAAGTTGGCA TTATAAAAAA GCATTGCTCA TCAATTTGTT GCAACGAACA GGTCACTATC AGTCAAAATA AAATCATTAT
  • Table 3 provides the sequences of the regions surrounding the core region for the wild type att sites ( ⁇ ttBO, P0, R0, and L0) as well as a variety of other suitable recombination sites. Those skilled in the art will appreciated that the remainder of the site may be the same as the corresponding site (B, P, L, or R) listed above.
  • recombination sites having unique specificity are known to those skilled in the art and may be used to practice the present invention.
  • Corresponding recombination proteins for these systems may be used in accordance with the invention with the indicated recombination sites.
  • Other systems providing recombination sites and recombination proteins for use in the invention include the FLP/FRT system from Saccharomyces cerevisiae, the resolvase family (e.g., ⁇ , TndX, TnpX, Tn3 resolvase, Hin, Hjc, Gin, SpCCEl, Par A, and Cin), and IS231 and other Bacillus thuringiensis transposable elements.
  • Other suitable recombination systems for use in the present invention include the XerC and XerD recombinases and the psi, dif and cer recombination sites in E. coli.
  • Other suitable recombination sites may be found in United States patent no. 5,851,808 issued to Elledge and Liu which is specifically incorporated herein by reference. Construction of Nucleic Acid Molecules
  • nucleic acid molecules of the invention are prepared as follows.
  • a first nucleic acid molecule comprising a sequence of interest may be prepared by methods known to those skilled in the art, such as by conventional cloning, recombinational cloning, topoisomerase-mediated cloning, PCR or other amplification technique, solid phase synthesis, etc.
  • a first nucleic acid molecule may comprise one or more recombination sites in addition to a sequence of interest.
  • One or more recombination sites may be located at any position in the first nucleic acid molecule and may be located upstream (i.e., in the 5'-direction of the sense strand) of the sequence of interest.
  • a sequence of interest may be encode one or more polypeptides or may code for an RNA molecule (e.g., a an antisense RNA, an siRNA, etc.).
  • at least one recombination site may be located adjacent to and/or upstream ofthe first base ofthe sequence of interest.
  • a second nucleic acid molecule comprising the sequence of one or more transcriptional regulatory sequences may be prepared using methods known to those skilled in the art.
  • a second nucleic acid molecule may also comprise one or more recombination sites and/or one or more transcriptional regulatory sequences.
  • Recombination sites may be located at any position in the second nucleic acid molecule.
  • a second nucleic acid molecule may comprise a transcriptional regulatory sequence adjacent to a recombination site. The recombination site may be located downstream (i.e., in the 3'-direction) of the transcriptional regulatory sequence.
  • Methods of construction of nucleic acid molecules of the invention may include a recombination reaction.
  • a first nucleic acid molecule having at least one recombination site may be recombined with a second nucleic acid molecule having at least one recombination site.
  • the first nucleic acid molecule may have a sequence encoding a nucleic acid molecule and/or protein of interest adjacent to and downstream of a recombination site.
  • the first nucleic acid molecule may be contacted with a second nucleic acid molecule optionally having a recombination site located adjacent to and downstream of a transcriptional regulatory sequence.
  • the first and the second nucleic acid molecules may be contacted under conditions sufficient to cause recombination between the recombination sites on the first and second nucleic acid molecules resulting in the production of a third nucleic acid molecule.
  • the coding sequence from the first nucleic acid molecule may be operably linked to the transcriptional regulatory sequence (when present) from the second nucleic acid molecule.
  • the first and the second nucleic acid molecules may be prepared by any technique known to those skilled in the art.
  • the first nucleic acid molecule may be a vector and the sequence encoding the protein of interest may be flanked by recombination sites.
  • suitable nucleic acid molecules that may serve as first nucleic acid molecules are sequences of interest in Entry Vectors available from Invitrogen Corporation, Carlsbad, CA, such as the clones in the ULTIMATETM ORF Collection, also available from Invitrogen Corporation, Carlsbad, CA.
  • a second nucleic acid molecule of the present invention may be a vector, for example, a Destination Vector compatible with the GATEWAYTM recombinational cloning technology of Invitrogen Corporation, Carlsbad, CA.
  • a second nucleic acid molecule may be a linear molecule.
  • a closed circular molecule e.g., a plasmid
  • a linear nucleic acid molecule to be used as a second nucleic acid molecule according to the present invention may be prepared by other techniques known in the art (e.g., PCR, chemical synthesis, etc.).
  • suitable second nucleic acid molecules include, but are not limited to, the T7- based pEXPl-DEST and pEXP2-DEST vectors supplied with the Expressway In Vitro Protein Synthesis System, Invitrogen Corporation, Carlsbad, CA, catalog nos. K9600-01, K9600-02, K9605-01, and K9605-02.
  • a schematic representation of the recombination region from these vectors after insertion of a sequence encoding a protein of interest is provided in Figure 2.
  • a third nucleic acid molecule produced by the methods of the invention may comprise a promoter recognized by T7 polymerase and additional sequences that facilitate in vitro transcription of a nucleic acid molecule corresponding to the third nucleic acid molecule and/or translation of the corresponding nucleic acid molecule.
  • the additional sequences may have been present on either the first or the second nucleic acid molecule or may have been partially contained on both.
  • a third nucleic acid molecule may comprise a T7 promoter, an initiation codon, and a prokaryotic Shine-Dalgarno ribosome binding site (RBS) upstream of the sequence encoding a protein of interest.
  • RBS prokaryotic Shine-Dalgarno ribosome binding site
  • sequence encoding the protein of interest may preferably comprise a stop codon.
  • additional sequences include, but are not limited to, purification tag sequences (e.g., six histidines), epitopes (e.g., V5 epitope) and the like.
  • Figure 3 provides a schematic representation of a suitable arrangement of T7 promoter and additional sequences for use in the present invention.
  • Third nucleic acid molecules may also comprise a sequence upstream ofthe T7 promoter containing a minimum of 6-10 nucleotides (nt) for efficient promoter binding. This sequence need not be specific.
  • Third nucleic acid molecules may also comprise a sequence following the T7 promoter containing a minimum of 15-20 nt that forms a potential stem-and-loop structure as described by Studier et al, 1990, Meth. Enzymol 185, 60-89.
  • Third nucleic acid molecules may also comprise a sequence of 9-11 nt between the RBS and the ATG initiation codon for optimal translation efficiency ofthe protein of interest. This sequence need not be specific.
  • Third nucleic acid molecules may also comprise a T7 terminator located 4-100 nt downstream of the sequence encoding the protein of interest for efficient transcription termination and message stability.
  • a third nucleic acid molecule produced as described above may be amplified using techniques well known in the art, for example, polymerase chain reaction (PCR).
  • PCR a well known DNA amplification technique, is a process by which DNA polymerase and deoxyribonucleoside triphosphates are used to amplify a target DNA template.
  • two primers one complementary to the 3' termini (or near the 3 '-terminus) of the first strand of the DNA molecule to be amplified, and a second primer complementary to the 3' termini (or near the 3 '-terminus) of the second strand of the DNA molecule to be amplified, are hybridized to their respective DNA strands.
  • DNA polymerase in the presence of deoxyribonucleoside triphosphates, allows synthesis of a third DNA strand complementary to the first strand and a fourth DNA strand complementary to the second strand ofthe DNA molecule to be amplified. This synthesis results in two double stranded DNA molecules.
  • double stranded DNA molecules may then be used to provide DNA templates for synthesis of additional DNA molecules by providing a DNA polymerase, primers, and deoxyribonucleoside triphosphates.
  • the additional synthesis is carried out by "cycling" the original reaction (with excess primers and deoxyribonucleoside triphosphates) allowing multiple denaturing and synthesis steps.
  • DNA polymerases including thermostable DNA polymerases
  • DNA polymerases include, but are not limited to, Taq DNA polymerase, Tne DNA polymerase, Tma DNA polymerase, Pfu DNA polymerase, Tfl DNA polymerase, Tth DNA polymerase, Tbr DNA polymerase, Pwo DNA polymerase, Bst DNA polymerase, Bca DNA polymerase, VENT DNA polymerase, T7 DNA polymerase, T5 DNA polymerase, DNA polymerase III, Klenow fragment DNA polymerase, Stoffel fragment DNA polymerase, and mutants, fragments or derivatives thereof.
  • SDA Strand Displacement Amplification
  • NASBA Nucleic Acid Sequence- Based Amplification
  • the present invention provides methods of amplifying one or more nucleic acid molecules. Such methods may comprise: (a) recombining one or more first nucleic acid molecules and one or more second nucleic acid molecules to form one or more third nucleic acid molecules;
  • a linear second nucleic acid molecule may be used and primer sites selected such that extension of each of the primers is away from the binding site of the other primer.
  • sites may be selected near the ends ofthe second nucleic acid molecule and the direction of extension may be toward the near end.
  • each primer is extended off the end of the second nucleic acid molecule and no amplification product is produced.
  • one or more third nucleic acid molecules may be double stranded nucleic acid molecules and such amplification methods may comprise:
  • such conditions according to the invention may include one or more nucleotides, one or more buffers or buffering salts, one or more primers, one or more cofactors, and/or one or more additional polypeptides having a nucleotide polymerase activity.
  • Those skilled in the art can readily optimize the conditions for conducting the recombination reactions described above without the use of undue experimentation, based on the guidance provided herein and available in the art (see, e.g., U.S. Patent Nos. 5,888,732 and 6,277,608, which are specifically incorporated herein by reference in their entireties).
  • a typical reaction from about 50 ng to about 1000 ng of a second nucleic acid molecule may be contacted with a first nucleic acid molecule under suitable reaction conditions.
  • Each nucleic acid molecule may be present in a molar ratio of from about 25:1 to about 1 :25 first nucleic acid molecule:second nucleic acid molecule.
  • a first nucleic acid molecule may be present at a molar ratio of from about 10:1 to 1:10 first nucleic acid molecule:second nucleic acid molecule. In a preferred embodiment, each nucleic acid molecule may be present at a molar ratio of about 1 :1 first nucleic acid molecule: second nucleic acid molecule.
  • the nucleic acid molecules may be dissolved in an aqueous buffer and added to the reaction mixture.
  • One suitable set of conditions is 4 ⁇ l CLONASETM enzyme mixture (e.g., Invitrogen Corporation, Cat. Nos. 11791- 019 and 11789-013), 4 ⁇ l 5X reaction buffer and nucleic acid and water to a final volume of 20 ⁇ l. This will typically result in the inclusion of about 200 ng of Int and about 80 ng of IHF in a 20 ⁇ l BP reaction and about 150 ng Int, about 25 ng IHF and about 30 ng Xis in a 20 ⁇ l LR reaction.
  • Proteins for conducting an LR reaction may be stored in a suitable buffer, for example, LR Storage Buffer, which may comprise about 50 mM Tris at about pH 7.5, about 50 mM NaCl, about 0.25 mM EDTA, about 2.5 mM Spermidine, and about 0.2 mg/ml BSA.
  • LR Storage Buffer may comprise about 50 mM Tris at about pH 7.5, about 50 mM NaCl, about 0.25 mM EDTA, about 2.5 mM Spermidine, and about 0.2 mg/ml BSA.
  • proteins for an LR reaction may be stored at a concentration of about 37.5 ng/ ⁇ l INT, 10 ng/ ⁇ l IHF and 15 ng/ ⁇ l XIS.
  • Proteins for conducting a BP reaction may be stored in a suitable buffer, for example, BP Storage Buffer, which may comprise about 25 mM Tris at about pH 7.5, about 22 mM NaCl, about 5 mM EDTA, about 5 mM Spermidine, about 1 mg/ml BSA, and about 0.0025% Triton X-100.
  • BP Storage Buffer may comprise about 25 mM Tris at about pH 7.5, about 22 mM NaCl, about 5 mM EDTA, about 5 mM Spermidine, about 1 mg/ml BSA, and about 0.0025% Triton X-100.
  • proteins for an BP reaction may be stored at a concentration of about 37.5 ng/ ⁇ l INT and 20 ng/ ⁇ l IHF.
  • enzymatic activity may vary in different preparations of enzymes. The amounts suggested above may be modified to adjust for the amount of activity in any specific preparation of enzymes.
  • a suitable 5X reaction buffer for conducting recombination reactions may comprise 100 mM Tris pH 7.5, 88 mM NaCl, 20 mM EDTA, 20 mM Spermidine, and 4 mg/ml BSA.
  • the final buffer concentrations may be 20 mM Tris pH 7.5, 17.6 mM NaCl, 4 mM EDTA, 4 mM Spermidine, and 0.8 mg/ml BSA.
  • the final reaction mixture may incorporate additional components added with the reagents used to prepare the mixture, for example, a BP reaction may include 0.005% Triton X-100 incorporated from the BP ClonaseTM.
  • the final reaction mixture may include about 50 mM Tris HCI, pH 7.5, about 1 mM EDTA, about 1 mg/ml BSA, about 75 mM NaCl and about 7.5 mM spermidine in addition to recombination enzymes and the nucleic acids to be combined.
  • the final reaction mixture may include about 25 mM Tris HCI, pH 7.5, about 5 mM EDTA, about 1 mg/ml bovine serum albumin (BSA), about 22 mM NaCl, and about 5 mM spermidine.
  • BSA bovine serum albumin
  • the final reaction mixture may include about 40 mM Tris HCI, pH 7.5, about 1 mM EDTA, about 1 mg/ml BSA, about 64 mM NaCl and about 8 mM spermidine in addition to recombination enzymes and the nucleic acids to be combined.
  • the reaction conditions may be varied somewhat without departing from the invention.
  • the pH of the reaction may be varied from about 7.0 to about 8.0; the concentration of buffer may be varied from about 25 mM to about 100 mM; the concentration of EDTA may be varied from about 0.5 mM to about 2 mM; the concentration of NaCl may be varied from about 25 mM to about 150 mM; and the concentration of BSA may be varied from 0.5 mg/ml to about 5 mg/ml.
  • the final reaction mixture may include about 25 mM Tris HCI, pH 7.5, about 5 mM EDTA, about 1 mg/ml bovine serum albumin (BSA), about 22 mM NaCl, about 5 mM spermidine and about 0.005% detergent (e.g., Triton X-100).
  • BSA bovine serum albumin
  • expression of a protein using the methods of the invention may comprise contacting a nucleic acid molecule (e.g., a template such as a third nucleic acid molecule as described above) with a cell extract under conditions sufficient to cause the expression of the protein.
  • the general system includes a nucleic acid molecule (e.g., a third nucleic acid molecule as described above) that functions as a template and encodes one or more desired proteins.
  • the desired protein(s) can be any polymer of amino acids encodable by a nucleic acid template to produce a protein molecule.
  • the protein can be further processed (e.g., modified, purified, etc) coincident with or after synthesis.
  • a second amino acid may be a normally occurring amino acid (e.g., Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, He, Leu, Lys, Met, Phe, Pro, Ser, Thr, Tip, Tyr, or Val).
  • codons in an mRNA that normally encode a first amino acid may result in incorporation into a polypeptide of a second amino acid.
  • a second amino acid may be a normally occurring amino acid (e.g., Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, He, Leu, Lys, Met, Phe, Pro, Ser, Thr, Tip, Tyr, or Val).
  • a second amino acid may be an unconventional or unnatural amino acids (e.g., detectably labeled amino acids). This may be accomplished, for example, by including tRNA molecules charged with molecules not normally incorporated into polypeptides by cells (see, for example, United States patent nos. 5,536,652 and 5,370,995, which are specifically incorporated herein by reference).
  • a template nucleic acid molecule produced as described above may comprise one or more sequences that function as promoters when contacted with a DNA-dependent RNA polymerase enzyme.
  • a template nucleic acid molecule comprising a promoter may be contacted with a polypeptide having DNA- dependent RNA polymerase activity under conditions causing the synthesis of one or more RNA molecules corresponding to the sequence of the template nucleic acid molecule encoding one or more protein of interest.
  • Suitable conditions may include one or more nucleotides (e.g., ribonucleotides), one or more buffers, one or more metal cofactors (e.g., Mg 2+ ), one or more nuclease inhibitors (e.g., RNase inhibitors) and the like.
  • the RNA molecules produced may be purified using standard techniques.
  • the reaction mixture containing the RNA molecules may be added directly to a protein synthesis reaction, which may comprise cell extract, as described in more detail below.
  • a template nucleic acid molecule may be contacted with the polypeptide having DNA-dependent RNA polymerase activity in the presence of all the necessary components to synthesis a protein and the RNA produced may be directly translated into protein without any further manipulation, i.e., a coupled in vitro transcription/translation reaction (IVTT) may be performed.
  • IVTT coupled in vitro transcription/translation reaction
  • Extracts to be used for the synthesis of proteins can be made from any suitable cells.
  • Suitable cells are those that have components for protein and/or nucleic acid synthesis, optionally produced under selected growth conditions or with modification(s) and/or mutation(s) that inactivate or reduce or substantially reduce unwanted properties detrimental to in vitro synthesis (e.g., nuclease activities, phosphatase activities, etc.).
  • Cells from which an extract may be prepared and used according to the invention include, but are not limited to, bacterial cells, fungal and yeast cells, plant cells and animal cells.
  • Preferred bacterial cells include, for example, gram positive and gram negative bacteria, and especially Escherichia spp. cells particularly E. coli cells and most particularly E.
  • coli strains DH10B, Stbl2, DH5 ⁇ , DB3, DB3.1 preferably E. coli LIBRARY EFFICIENCY ® DB3.1TM Competent Cells; Invitrogen Corporation, Carlsbad, CA
  • DB4 and DB5 see U.S. Application No. 09/518,188, filed on March 2, 2000, the disclosure of which are incorporated by reference herein in their entireties
  • Other preferred bacterial host cells include, but are not limited to, Bacillus spp. cells (particularly B. subtilis and B.
  • Preferred animal host cells include insect cells (especially, Drosophila melanogaster cells and more especially S2 cells, Spodoptera frugiperda Sf9 and Sf21 cells and Trichoplusa High-Five cells), nematode cells (particularly C.
  • elegans cells avian cells
  • amphibian cells particularly Xenopus laevis cells
  • reptilian cells and mammalian cells (most particularly NLH3T3, CHO, COS, C127, VERO, BHK, HeLa, 293, Per-C6, Bowes melanoma, and human, rabbit, mouse, rat, hamster, pig, bovine and gerbil cells generally).
  • Preferred fungal and yeast host cells include Aspergillus spp. cells, Saccharomyces cerevisiae cells and Pichia pastoris cells.
  • Plant cells are exemplified by protoplasts, tobacco, potato and other tuberous plants, grasses including maize, cotton and other fibrous plants, annuals and perennials, monocots and dicots, and especially plant cells that can be transformed and/or grown in culture.
  • the cell extract can be supplemented to provide components not present or not present in sufficient quantities after extraction.
  • the extract can be prepared by any method used in the art that maintains the integrity of the transcription/translation system or, if the process damages one or more component necessary for any stage of transcription/translation, the damaged component can be replaced or substituted for after the extract preparation.
  • Extracts can be prepared according to the method of Zubay (1973),
  • extracts may be supplemented with one or more polymerase enzymes, for example, T7 polymerase, SP6 polymerase and the like.
  • polymerase enzymes for example, T7 polymerase, SP6 polymerase and the like.
  • Other methods of preparing and/or supplementing extracts suitable for IVTT reactions are known in the art, for example, as disclosed in WO 02/072890 and published United States patent application 2002-0168706 Al.
  • Other embodiments may utilize extracts prepared from eukaryotic organisms, for example, wheat germ.
  • eukaryotic systems are well known in the art (see, for example, United States Patent nos. 5,665,563 and 5,492,817).
  • Suitable extracts of wheat germ can be prepared by a methods known in the art (see, for example, Roberts, B. E. and Paterson, B. M., Proc. Natl Acad. Sci. USA Vol. 70, No. 8, pp. 2330-2334, (1973); and C. W., et al. Meth. Enzymol. Vol. 101, p. 635, (1983)).
  • wheat germ extract may be prepared by grinding wheat germ in an extraction buffer, followed by centrifugation to remove cell debris. The supernatant is then separated by chromatography from endogenous amino acids and plant pigments that are inhibitory to translation.
  • the extract may also be treated with one or more nuclease enzymes (e.g., micrococcal nuclease) to destroy endogenous mRNA, thereby reducing the production of undesired proteins.
  • the extract may contain and/or may be supplemented with the cellular components necessary for protein synthesis, such as tRNA, rRNA and initiation, elongation, and termination factors.
  • the extract may be supplemented with one or more components, (e.g., energy generating systems, metal ions, buffers, etc).
  • Suitable eukaryotic cell extracts are commercially available, for example, the TNT® Coupled Wheat Germ Extract System and TNT® Coupled Reticulocyte Lysate System, from Promega Corporation, Madison, Wl; and the Retic Lysate IVTTM and Wheat Germ INTTM, from Ambion, Inc. Austin, TX.
  • TNT® Coupled Wheat Germ Extract System and TNT® Coupled Reticulocyte Lysate System from Promega Corporation, Madison, Wl
  • Retic Lysate IVTTM and Wheat Germ INTTM from Ambion, Inc. Austin, TX.
  • Other suitable systems and protocols are described in Methods in Molecular Biology, Vol. 37: In Vitro Transcription and Translation Protocols,
  • a non-limiting example of a suitable set of reaction conditions for conducting a transcription translation reaction is 57 mM Hepes/KOH pH 8.2, 230 mM K-glutamate, 1.2 mM ATP, 0.85 mM each of GTP, UTP, CTP, 30 mM PEP, 1.7 mM DTT, 12 mM Mg(OAc) 2 , 0.17 mg/ml E.
  • coli total RNA mixture 34 ⁇ g/ml folinic acid, 66 ⁇ g/ml T7 RNA polymerase, 1.25 mM each of amino acids, 14 mg/ml extract, 3 ⁇ l of 5 S-Met (15 ⁇ Ci/ ⁇ l) if labeled proteins are desired and various amount of DNA template.
  • reaction buffer may comprise all the required amino acids and an ATP regenerating system (see, for example, Kim et al, (1996) Eur. J. Biochem. 239, 881-886; Lesley, et al. (1991) J. Biol. Chem. 266, 2632-2638; and Pratt (1984) Transcription and Translation (Oxford: S. . LRL Press).
  • concentrations of the various components of the incubation medium can be adjusted as is known in the art while still maintaining the synthetic function.
  • the pH can range from about 5.6 to about 8.8 or more preferably about 6.1 to 8.5 or even about 7.2 to 8.4 depending on the product to be synthesized.
  • the concentration of K-glutamate can be easily varied between about 80 mM and 320 mM or more preferably about 120 to 280 mM or even about 180 to 250 mM; the concentration of ATP can be varied from about 0.1 to 3.0 mM or more preferably about 0.3 to 2.0 mM or even about 0.8 to 1.5 mM; the concentrations of GTP, UTP CTP and TTP can vary from about 0 or 0.1 to 2 mM or more preferably about 0.4 to 1.2 mM or even about 0.7 to 1.0 mM; PEP can vary from about 10 to 60 mM or more preferably about 20 to 50 mM or even about 25 to 40 mM; DTT can vary from about 0.5 to 3.0 mM or more preferably about 1.0 to 2.5 mM or even about 1.5 to 2.0 mM; magnesium can vary from about 7.5 to 20 mM or more preferably about 10 to 15 mM or even about 11.5 to 14 mM; total RNA mixture can vary from about 0.1 to
  • a third nucleic acid molecule as described above may be purified and resuspended in a suitable buffer (e.g., 10 mM Tris, 1 mM EDTA, pH 7.0 to 8.0) or water such that the final concentration is greater than 500 ng/ ⁇ l. It is desirable to ensure that the purified third nucleic acid molecule is not contaminated with ethanol, salt, or RNases.
  • a suitable buffer e.g. 10 mM Tris, 1 mM EDTA, pH 7.0 to 8.0
  • water such that the final concentration is greater than 500 ng/ ⁇ l. It is desirable to ensure that the purified third nucleic acid molecule is not contaminated with ethanol, salt, or RNases.
  • a third nucleic acid molecule may be purified using techniques well known in the art, for example, commercial DNA purification kits (e.g., the S.N.A.P.TM MiniPrep Kit (Catalog no. Kl 900-01 available from Invitrogen Corporation, Carlsbad, CA).
  • S.N.A.P.TM MiniPrep Kit Catalog no. Kl 900-01 available from Invitrogen Corporation, Carlsbad, CA.
  • For protocols to purify DNA refer to published reference sources (Ausubel et al, (1994) Current Protocols in Molecular Biology.; Sambrook et al, (1989) Molecular Cloning: A Laboratory Manual, Second Edition (Plainview, New York: Cold Spring Harbor Laboratory Press).
  • a third nucleic acid molecule should not be purified by gel elctrophoresis as purified DNA solution obtained from agarose gels significantly inhibits the protein synthesis reaction.
  • ammonium acetate is not recommended for use in DNA precipitation as any residual contamination may inhibit translation.
  • Sodium acetate may be used in DNA precipitation. Care should be taken to avoid contamination of the solution containing the third nucleic acid molecule with nucleases, e.g., RNases. Standard techniques, e.g., wearing gloves and using RNase-free reagents can be employed. Purified third nucleic acid molecule should be free of excess ethanol or salt as both can inhibit translation. When ethanol precipitation is used to purify DNA, DNA should be carefully washed with 70% ethanol to remove excess salt and dried.
  • a third nucleic acid molecule may be mixed with an appropriate amount of cell extract, optionally supplemented with one or more ingredients (e.g., buffers, polymerases, amino acids, etc.) and incubated, for example, at 37 °C for from about 10 minutes to about 10 hours, from about 30 minutes to about 8 hours, from about 1 hour to about 8 hours, from about 1 hour to about 7 hours, from about 1 hour to about 6 hours, from about 1 hour to about 5 hours, from about 1 hour to about 4 hours, from about 1 hour to about 3 hours, or from about 1 hour to about 2 hours.
  • the reaction may be shaken, for example, using an Eppendorf Thermomixer (Fisher, Catalog no. 05-400-200) or other equivalent means.
  • the protein may be further processed (e.g., purified, analyzed etc.) using techniques known in the art. Suitable techniques include, but are not limited to, Coomassie -stained protein gel, Western blot, enzymatic assays, affinity purification (if affinity tag is present), and the like.
  • kits that may be used in conjunction with methods the invention.
  • Kits according to this aspect of the invention may comprise one or more containers, which may contain one or more components selected from the group consisting of one or more nucleic acid molecules, one or more primers, one or more polymerases, one or more recombination proteins (or other enzymes for carrying out the methods of the invention), one or more buffers, one or more detergents, one or more restriction endonucleases, one or more nucleotides, pyrophosphatase, and the like.
  • nucleic acid molecules can be used with the invention.
  • nucleic acid molecules that can be supplied in kits of the invention include those that contain or encode promoters, signal peptides, enhancers, repressors, suppressors, selection markers, transcription signals, translation signals, primer hybridization sites (e.g., for sequencing or PCR), recombination sites, restriction sites and polylinkers, sequences that encode domains and/or regions (e.g., 6 His tag) for the preparation of fusion proteins, and the like.
  • libraries can be supplied in kits of the invention. These libraries may be in the form of replicable nucleic acid molecules or they may comprise nucleic acid molecules that are not associated with an origin of replication.
  • nucleic acid molecules of libraries as well as other nucleic acid molecules that are not associated with an origin of replication, either could be inserted into other nucleic acid molecules that have an origin of replication or could be provided without being associated with an origin or replication in the kits of the invention.
  • libraries supplied in kits of the invention may be used as first nucleic acid molecules as described above and may comprise two components: (1) the nucleic acid molecules of these libraries and (2) 5' and/or 3' recombination sites.
  • the nucleic acid molecules of a library when supplied with 5' and/or 3' recombination sites, it will be possible to recombine these molecules with a second nucleic acid molecule as described above to produce a third nucleic acid molecule, using recombination reactions.
  • recombination sites can be attached to the nucleic acid molecules of the libraries before use (e.g., by the use of a ligase, which may also be supplied with the kit). In such cases, nucleic acid molecules that contain recombination sites or primers that can be used to generate recombination sites may be supplied with the kits.
  • Kits of the invention may further comprise containers containing one or more recombination proteins.
  • Suitable recombination proteins have been disclosed above and include, but are not limited to, Cre, Int, LHF, Xis, Flp, Fis, Hin, Gin, Cin, Tn3 resolvase, ⁇ C31, TndX, XerC, and XerD.
  • Kits of the invention can also comprise one or more primers. These primers will generally be designed to anneal to molecules having specific nucleotide sequences. For example, these primers can be designed for use in PCR to amplify a particular nucleic acid molecule. Further, primers supplied with kits of the invention can be sequencing primers designed to hybridize to vector sequences. Thus, such primers will generally be supplied as part of a kit for sequencing nucleic acid molecules that have been inserted into a vector.
  • One or more buffers may be supplied in kits of the invention. These buffers may be supplied at a working concentrations or may be supplied in concentrated form and then diluted to the working concentrations. These buffers will often contain salt, metal ions, co-factors, metal ion chelating agents, etc. for the enhancement of activities of the stabilization of either the buffer itself or molecules in the buffer. Further, these buffers may be supplied in dried or aqueous forms. When buffers are supplied in a dried form, they will generally be dissolved in water prior to use.
  • Kits of the invention may contain virtually any combination of the components set out above or described elsewhere herein, or other components known to those of ordinary skill to be useful in carrying out methods of the invention. As one skilled in the art would recognize, the components supplied with kits of the invention will vary with the intended use for the kits. Thus, kits may be designed to perform various functions set out in this application and the components of such kits will vary accordingly.
  • Kits of the invention may further comprise one or more pages of written instructions for carrying out the methods of the invention.
  • instructions may comprise methods steps necessary to carryout recombinational cloning of an ORF provided with recombination sites and a vector also comprising recombination sites and optionally further comprising one or more functional sequences (e.g., transcriptional regulatory sequences).
  • Instructions may comprise the steps necessary to carry out an in vitro transcription/translation reaction and/or an amplification reaction.
  • the present invention also provides a system and method of providing company products to a party outside of the company, for example, a system and method for providing a customer or a product distributor a product of the company such as a kit containing materials for practicing the methods of the invention and/or instructions for practicing such methods.
  • Figure 4 provides a schematic diagram of a product management system.
  • the blocks in Figure 4 can represent an intra-company organization, which may include departments in a single building or in different buildings, a computer program or suite of programs maintained by one or more computers, a group of employees, a computer I/O device such as a printer or fax machine, a third party entity or company that is otherwise unaffiliated with the company, or the like.
  • the product management system as shown in Figure 4 is exemplified by company 100, which receives input in the form of an order from a party outside of the company, e.g., distributor 150 or customer 140, to order department 126, or in the form of materials and parts 130 from a party outside of the company; and provides output in the form of a product delivered from shipping department 119 to distributor 150 or customer 140.
  • Company 100 system is organized to optimize receipt of orders and delivery of a products to a party outside of the company in a cost efficient manner, particularly instructions or a kit of the present invention, and to obtain payment for such product from the party in a timely manner.
  • materials and parts refers to items that are used to make a device, other component, or product, which generally is a device, other component, or product that company sells to a party outside of the company.
  • materials and parts include, for example, recombinases, starting nucleic acid molecules with one or more recombination sites, nucleic acid segments, nucleotides, host cells, polymerases, amino acids, buffers, paper, ink, reaction vessels, etc.
  • kits refer to items sold by the company.
  • Other components are exemplified by instructions, including instructions for the practice of the methods of the invention.
  • Other components also can be items that may be included in a kit, e.g., a kit product, for example, reagents for manipulating nucleic acid molecules (e.g., performing recombinational cloning).
  • Kits may include multiple marker molecules and instructions for use.
  • Reagents may include, for example, buffers, salts, cofactors and the like.
  • Other components may include host cell extracts. As such, it will be recognized that an item useful as materials and parts as defined herein further can be considered another component, which can be sold by the company.
  • kits which can contain instructions according to the present invention, and one or more nucleic acid molecules, one or more cell extracts, one or more enzymes (e.g., polymerases), one or more primers, one or more reagents, one or more buffers or buffer salts, and/or combinations thereof.
  • Nucleic acid molecules may be comprise one or more recombination sites.
  • company 100 includes manufacturing 110 and administration 120.
  • Devices 112 and other components 114 are produced in manufacturing 110, and can be stored separately therein such as in device storage 113 and other component storage 115, respectively, or can be further assembled and stored in product storage 117.
  • Materials and parts 130 can be provided to company 100 from an outside source and/or materials and parts 114 can be prepared in company, and used to produce devices 112 and other components 116, which, in turn, can be assembled and sold as a product.
  • Manufacturing 110 also includes shipping department 119, which, upon receiving input as to an order, can obtain products to be shipped from product storage 117 and forward the product to a party outside the company.
  • product storage 117 can be used to store instructions for practicing methods of the invention; or can store kits, which can contain at least one nucleic acid molecule, one or more polymerases, one or more primers and/or one or more cell extracts, and, optionally, instructions as disclosed herein; or can store a combination of such instructions and/or kits.
  • shipping department 119 can obtain from product storage 117 such kit for shipping, and can further obtain such instructions in a written form to include with the kit, and ship the kit and instructions to customer 140 (and providing input to billing department 124 that the product was shipped; or shipping department 119 can obtain from product storage 117 the kit for shipping, and can further provide the instructions to customer 140 in an electronic form, by accessing a database in company 100 that contains the instructions, and transmitting the instructions to customer 140 via the internet (not shown).
  • administration 120 includes order department 126, which receives input in the form of an order for a product from customer 140 or distributor 150.
  • Order department 126 then provides output in the form of instructions to shipping department 119 to fill the order, i.e., to forward products as requested to customer 140 or distributor 150.
  • Shipping department 119 in addition to filling the order, further provides input to billing department 124 in the form of confirmation of the products that have been shipped.
  • Billing department 124 then can provide output in the form of a bill to customer 140 or distributor 150 as appropriate, and can further receive input that the bill has been paid, or, if no such input is received, can further provide output to customer 140 or distributor 150 that such payment may be delinquent.
  • customer service department 122 can receive input from customer 140 and can provide output in the form of feedback or information to customer 140.
  • customer service 122 can receive input or provide output to any other component of company.
  • customer service department 122 can receive input from customer 140 indicating that an ordered product was not received, wherein customer service department 122 can provide output to shipping department 119 and or order department 126 and/or billing department 124 regarding the missing product, thus providing a means to assure customer 140 satisfaction.
  • Customer service department 122 also can receive input from customer 140 in the form of requested technical information, for example, for confirming that instructions of the invention can be applied to the particular need of customer 140, and can provide output to customer 140 in the form of a response to the requested technical information.
  • the components of company 100 are suitably configured to communicate with each other to facilitate the transfer of materials and parts, devices, other components, products, and information within company 100, ⁇ u ⁇ u u ⁇ mpany l ⁇ is rurtner suitably configured to receive input from or provide output to an outside party.
  • a physical path can be utilized to transfer products from product storage 117 to shipping department 119 upon receiving suitable input from order department 126.
  • Order department 126 in comparison, can be linked electronically with other components within company 100, for example, by a communication network such as an intranet, and can be further configured to receive input, for example, from customer 140 by a telephone network, by mail or other carrier service, or via the internet.
  • a direct electronic link such as a Tl line or a direct wireless connection also can be established, particularly within company 100 and, if desired, with distributor 150 or materials or parts 130 provider, or the like.
  • company 100 may comprise one or more data collection systems, including, for example, a customer data collection system, which can be realized as a personal computer, a computer network, a personal digital assistant (PDA), an audio recording medium, a document in which written entries are made, any suitable device capable of receiving data, or any combination of the foregoing.
  • Data collection systems can be used to gather data associated with a customer 140 or distributor 150, including, for example, a customer's shipping address and billing address, as well as more specific information such as the customer's ordering history and payment history, such data being useful, for example, to determine that a customer has made sufficient purchases to qualify for a discount on one or more future purchases.
  • Company 100 can utilize a number of software applications to provide components of company 100 with information or to provide a party outside of company access to one or more components of company 100, for example, access to order department 126 or customer service department 122.
  • Such software applications can comprise a communication network such as the Internet, a local area network, or an intranet.
  • customer 140 can access a suitable web site and/or a web server that cooperates with order department 126 such that customer 140 can provide input in the form of an order to order department 126.
  • order department 126 can communicate with customer 140 to confirm that the order has been received, and can further communicate with shipping department 119, providing input that products such as a kit of the invention, which contains, for example, one or more nucleic acid molecules having one or more recombination sites, one or more polymerases, one or more primers and/or one or more cell extracts and instructions for use, should be shipped to customer 140.
  • a kit of the invention which contains, for example, one or more nucleic acid molecules having one or more recombination sites, one or more polymerases, one or more primers and/or one or more cell extracts and instructions for use, should be shipped to customer 140.
  • the business of company 100 can proceed in an efficient manner.
  • billing department 124 and shipping department 119 can communicate with one another by way of respective computer systems.
  • computer system refers to general purpose computer systems such as network servers, laptop systems, desktop systems, handheld systems, personal digital assistants, computing kiosks, and the like.
  • distributor 150 can access a web site maintained by company 100 after establishing an online connection to the network, particularly to order department 126, and can provide input in the form of an order. If desired, a hard copy of an order placed with order department 126 can be printed from the web browser application resident at distributor 150.
  • the various software modules associated with the implementation of the present invention can be suitably loaded into the computer systems resident at company 100 and any party outside of company 100 as desired, or the software code can be stored on a computer-readable medium such as a floppy disk, magnetic tape, or an optical disk.
  • a server and web site maintained by company 100 can be configured to provide software downloads to remote users such as distributor 150, materials and parts 130, and the like.
  • the techniques of the present invention are carried out by code segments and instructions associated with the various process tasks described herein.
  • the present invention further includes methods for providing various aspects of a product (e.g., a kit and/or instructions of the invention), as well as information regarding various aspects of the invention, to parties such as the parties shown as customer 140 and distributor 150 in Figure 4.
  • a product e.g., a kit and/or instructions of the invention
  • parties such as the parties shown as customer 140 and distributor 150 in Figure 4.
  • methods for selling devices, products and methods of the invention to such parties are provided, as are methods related to those sales, including customer support, billing, product inventory management within the company, etc. Examples of such methods are shown in Figure 4, including, for example, wherein materials and parts 130 can be acquired from a source outside of company 100 (e.g., a supplier) and used to prepare devices used in preparing a composition or practicing a method of the invention, for example, kits, which can be maintained as an inventory in product storage 117.
  • devices 112 can be sold directly to a customer and/or distributor (not shown), or can be combined with one or more other components 116, and sold to a customer and/or distributor as the combined product.
  • the other components 116 can be obtained from a source outside of company 100 (materials and parts 130) or can be prepared within company 100 (materials and parts 114).
  • the term "commercial product" is used generally herein to refer an item sent to a party outside of the company (a customer, a distributor, etc.) and includes items such as devices 112, which can be sent to a party alone or as a component of a kit or the like. At the appropriate time, the product is removed from product storage
  • 117 for example, by shipping department 119, and sent to a requesting party such as customer 140 or distributor 150.
  • a requesting party such as customer 140 or distributor 150.
  • shipping occurs in response to the party placing an order, which is then forwarded the within the organization as exemplified in Figure 4, and results in the ordered product being sent to the party.
  • Data regarding shipment of the product to the party is transmitted further within the organization, for example, from shipping department 119 to billing department 124, which, in turn, can transmit a bill to the party, either with the product, or at a time after the product has been sent.
  • a bill can be sent in instances where the party has not paid for the product shipped within a certain period of time (e.g., within 30 days, within 45 days, within 60 days, within 90 days, within 120 days, within from 30 days to 120 days, within from 45 days to 120 days, within from 60 days to 120 days, within from 90 days to 120 days, within from 30 days to 90 days, within from 30 days to 60 days, within from 30 days to 45 days, within from 60 days to 90 days, etc.).
  • billing department 124 also is responsible for processing payment(s) made by the party.
  • customer service department 122 can receive an order from the party, and transmit the order to shipping department 119 (not shown), thus serving the functions exemplified in Figure 4 by order department 126 and the customer service department 122.
  • the methods of the invention also include providing technical service to parties using a product, particularly a kit of the invention. While such a function can be performed by individuals involved in product research and development, inquiries related to technical service generally are handled, routed, and/or directed by an administrative department of the organization (e.g., customer service department 122). Often communications related to technical service (e.g., solving problems related to use of the product or individual components of the product) require a two way exchange of information, as exemplified by arrows indicating pathways of communication between customer 150 and customer service department 122.
  • the invention includes methods (e.g., business methods) that involve (1) the production of products (e.g., nucleic acid and/or protein molecules, kits that contain instructions for performing methods of the invention, etc.); (2) receiving orders for these products; (3) sending the products to parties placing such orders; (4) sending bills to parties obliged to pay for products sent to such; and/or (5) receiving payment for products sent to parties.
  • products e.g., nucleic acid and/or protein molecules, kits that contain instructions for performing methods of the invention, etc.
  • receiving orders for these products e.g., nucleic acid and/or protein molecules, kits that contain instructions for performing methods of the invention, etc.
  • sending the products to parties placing such orders e.g., (4) sending bills to parties obliged to pay for products sent to such; and/or (5) receiving payment for products sent to parties.
  • methods comprise two or more of the following: (a) obtaining parts, materials, and/or components from a supplier; (b) preparing one or more first products (e.g., one or more nucleic acid and/or protein molecules); (c) storing the one or more first products of (b); (d) combining the one or more first products of (b) with one or more other components to form one or more second products (e.g., a kit); (e) storing the one or more first products of (b) or one or more second products of (d); (f) obtaining an order a first product of (b) or a second product of (d); (g) shipping either the first product of (b) or the second product of (d) to the party that placed the order of (f); (h) tracking data regarding to the amount of money owed by the party to which the product is shipped in (g); (i) sending a bill to the party to which the product is shipped in (g); (j) obtaining payment for the product shipped in (g)
  • first products
  • the present invention also provides a system and method for providing information as to availability of a product (e.g., a device product, a kit product, and the like) to parties having potential interest in the availability of the kit product.
  • a product e.g., a device product, a kit product, and the like
  • Such a method of the invention which encompasses a method of advertising to the general or a specified public, the availability of the product, particularly a product comprising instructions and/or a kit of the present invention, can be performed, for example, by transmitting product description data to an output source, for example, an advertiser; further transmitting to the output source instructions to publish the product information data in media accessible to the potential interested parties; and detecting publication of the data in the media, thereby providing information as to availability of the product to parties having potential interest in the availability of the product.
  • the present invention provides methods for advertising and/or marketing devices, products, and/or methods of the invention, such methods providing the advantage of inducing and/or increasing the sales of such devices, products, and/or methods.
  • advertising and/or marketing methods of the invention include those in which technical specifications and/or descriptions of devices and/or products; methods of using the devices and/or products; and/or instructions for practicing the methods and/or using the devices and/or products are presented to potential interested parties, particularly potential purchasers of the product such as customers, distributors, and the like.
  • the advertising and/or marketing methods involve presenting such information in a tangible form or in an intangible to the potential interested parties.
  • tangible form means a form that cannot be physically handled and includes, for example, electronic media (e.g., e-mail, internet web pages, etc.), broadcasts (e.g., television, radio, etc.), and direct contacts (e.g., telephone calls between individuals, between automated machines and individuals, between machines, etc.); whereas the term "tangible form” means a form that can be physically handled.
  • electronic media e.g., e-mail, internet web pages, etc.
  • broadcasts e.g., television, radio, etc.
  • direct contacts e.g., telephone calls between individuals, between automated machines and individuals, between machines, etc.
  • tangible form means a form that can be physically handled.
  • the blocks in Figure 5 can represent an intra-company organization, which can include departments in a single building or in different buildings, a computer program or suite of programs maintained by one or more computers, a group of employees, a computer I/O device such as a printer or fax machine, a third party entity or company that is otherwise unaffiliated with the company, or the like.
  • the information providing management system as shown in Figure 5 is exemplified by company 200, which makes, purchases, or otherwise makes available devices and methods 210 that alone, or in combination, provide products 220, for example, instructions, devices and/or kits of the present invention, that company 200 wishes to sell to interested parties.
  • product descriptions 230 are made, providing information that would lead potential users to believe that products 220 can be useful to user.
  • product descriptions 230 is provided to advertising agency 240, which can be an entity separate from company 200, or to advertising department 260, which can be an entity related to company 200, for example, a subsidiary.
  • advertisement 250 is generated and is provided to media accessible to potential purchasers of products 260, whom may then contact company 200 to purchase products 220.
  • product descriptions 230 can be in a tangible form such as written descriptions, which can be delivered (e.g., mailed, sent by courier, etc) to advertising agency 240 and/or advertising department 250, or can be in an intangible form such as entered into and stored in a database (e.g., on a computer, in an electronic media, etc.) and transmitted to advertising agency 240 and/or advertising department 250 over a telephone line, Tl line, wireless network, or the like.
  • advertisement 250 can be a tangible or intangible form such that it conveniently and effectively can be provided to potential parties of interest (e.g., potential purchasers of product 260).
  • advertisement 250 can be provided in printed form as flyers (e.g., at a meeting or other congregation of potential interested parties) or as printed pages (or portions thereof) in magazines known to be read by the potential interested parties (e.g., trade magazines, journals, newspapers, etc.).
  • advertisement 250 can be provided in the form of directed mailing of computer media containing the advertisement (e.g., CDs, DVDs, floppy discs, etc.) or of e-mail (i.e., mail or e-mail that is sent only to selected parties, for example, parties known to members of an organization that includes or is likely to include potential users of products 220); of web pages (e.g., on a website provided by company 200, or having links to the company 200 website); or of pop-up or pop-under ads on web pages known to be visited by potential purchaser of products 260, and the like.
  • Potential purchasers of products 260 upon being apprised of the availability of the products 220, for example, the kits of the present invention, then can contact company 200 and, if so desired, can order said products 220 for company 200 (see Figure 4)
  • a cell-free protein synthesis system for example, one that uses a wheat germ extract, may be used.
  • Cell-free protein synthesis systems that use wheat germ extracts duplicate eukaryotic protein synthesis systems in vitro, and have high protein synthesis functionality.
  • the expression of proteins that affect cell physiology is also possible with such a system, and high-throughput synthesis is easy due to the in vitro test system.
  • proteins may be synthesized using the following procedures: (1) construction of expression clones through an LR reaction, (2) amplification of template DNA through PCR, (3) in vitro mRNA synthesis, and (4) protein synthesis using a wheat germ cell-free protein synthesis system.
  • the methods of the invention disclosed herein enable in vitro performance of all processes from the construction of expression clones to protein synthesis.
  • the methods described herein permit the completion of the entire process, expression clone through protein synthesis, more rapidly that was previously possible.
  • operations may be conducted in a high-throughput method (e.g., using a 96-well plate format).
  • the present invention overcomes many of the problems associated with prior art methodologies, such as those that require introduction of a template containing nucleic acid molecule into a host cell.
  • prior art methodologies such as those that require introduction of a template containing nucleic acid molecule into a host cell.
  • experiments requiring the introduction of genetic material into a cell are classified as recombinant DNA experiments and are subject to governmental restrictions.
  • Previously known methods are, therefore, subject to these restrictions making it difficult for scientists and students.
  • Methods of the present invention do not require introduction of genetic material into a host cell and, therefore, are not subject to the same restrictions.
  • FIG. 6 A provides a timeline of the production of a polypeptide.
  • Fig. 6B shows a schematic representation of the reactions performed in one embodiment of the present invention.
  • an entry clone is reacted with a destination vector in an LR reaction (shown as (2) in Fig. 6B), the reaction product is transformed into E. coli, plasmid is purified and the purified plasmid is used as a template in an amplification reaction.
  • a destination vector is first digested with a restriction enzyme (shown as (1) in Fig. 6B), an LR reaction is conducted (2), a PCR reaction is conducted on the LR reaction product (3), the PCR product (4) may be used in an in vitro transcription reaction (5), and the RNA transcripts thus produced may be used in an in vitro translation reaction (6).
  • the PCR product may be used in a coupled transcription/translation reaction.
  • the plasmid may be used as an amplification (e.g., PCR) template.
  • amplification e.g., PCR
  • Use of a host cell introduces several potential problems. For example, proteins having toxicity or proteins with high physiological activity adversely affect the growth of E. coli, which can result in variation in plasmid yield. This variable yield makes it difficult to handle such clones in a high throughput system for protein expression.
  • plasmid DNA prepared from a host cell may contain RNase contamination resulting in the degradation of mRNA produced from the template. The present invention obviates these difficulties.
  • first nucleic acid molecules comprising a sequence of interest encoding a polypeptide and comprising one or more recombination sites are prepared.
  • a first nucleic acid molecule is shown as an Entry Clone for use in a GATEWAYTM LR reaction.
  • the sequence of interest (indicated as Gene(ORF) in Figure 7) is flanked with recombination sites (shown as ⁇ ttLl and ⁇ ttL2 in Figure 7).
  • the first nucleic acid molecule is shown as a plasmid with an origin of replication (ori) and a selectable marker (e.g., the kanamycin resistance gene, Km).
  • a second nucleic acid molecule comprising at least one transcriptional regulatory sequence and one or more recombination sites is also prepared.
  • a second nucleic acid molecule is shown as a plasmid comprising a transcriptional regulatory sequence (e.g., the SP6 promoter) and an additional sequence that affects transcription and/or translation (e.g., the ⁇ translation enhancer sequence).
  • the sequence of vector pEU3'His is provided in Table 4. This vector contain the ⁇ sequence, a translational enhancer from 5 '-untranslated sequence of tobacco mosaic virus.
  • the ⁇ sequence is a 71-mer having the following sequence:
  • the first and second nucleic acid molecule may be contacted under conditions causing recombination of the first and second nucleic acid molecules to produce a third nucleic acid molecule in which the sequence of interest and transcriptional regulatory sequence are operably linked.
  • the third nucleic acid molecule is indicated as an Expression Clone.
  • the third nucleic acid molecule shown in Figure 7 is a plasmid resulting from the recombination of the ⁇ ttLl and ⁇ ttL2 sites in the first nucleic acid molecule and the ⁇ ttRl and ⁇ ttR2 sites in the second nucleic acid molecule. It is not necessary that recombination occur in both sets of sites.
  • a third nucleic acid molecule having a transcriptional regulatory sequence operably linked to a sequence of interest will be produced.
  • other second nucleic acid molecules having only a single recombination site may be used in the practice of the invention.
  • both the first and second nucleic acid molecules are shown as plasmids in Figure 7, either or both may not be a plasmid (e.g., may be a PCR fragment, restriction fragment, synthetic molecule, etc.).
  • a linear nucleic acid molecule as a second nucleic acid molecule.
  • linearization of the second nucleic acid molecule may reduce background in the amplification reaction.
  • the destination vector pEU3'His for construction of an expression vector for use in a wheat germ cell-free protein synthesis system
  • BgtR and Pstl restriction enzyme sites indicated
  • a third nucleic acid molecule (e.g., the expression clone of Figure 7) is created.
  • the third nucleic acid molecule may be amplified, for example, using primers that flank the recombination region of the second nucleic acid molecule.
  • a GATEWAYTM LR reaction with GATEWAYTM-adapted entry clones and a destination vector adapted for IVTT was conducted, resulting in the construction of expression clones into which the targeted gene ORF was inserted through site- specific recombination reactions between ⁇ ttLl and ⁇ ttRl, and ⁇ ttL2 and ⁇ ttR2.
  • the GATEWAYTM LR reaction was performed in a 5 ⁇ l reaction volume using the restriction digested destination vector pEU3'His reaction and various entry clones for 3 hours.
  • Entry clones are pDONR201 -based plasmids (Invitrogen Corporation, Carlsbad, CA) harboring ORFs of EGFP and human full-length cDNA. Methods of the invention were tested on about 4000 clones. The sequences of some ofthe clones tested are provided in Tables 5-8.
  • amplification reaction may be conducted using the reaction product of the LR reaction described above as a template.
  • the amplification may be conducted in a 50 ⁇ l reaction mixture using KOD Dash as a polymerase and 1 ⁇ l of the LR reaction as a template and the following primers
  • An amplified third nucleic acid molecule may be used as a template for in vitro transcription.
  • the transcription reaction may be performed in a 50 ⁇ l reaction mixture using an appropriate polymerase.
  • the transcription reaction was conducted using SP6 polymerase at 37 °C for 4 hours.
  • a typical mRNA synthesis reaction may contain: 5 x TB Buffer 10.0 ⁇ l
  • the transcription reaction may be conducted in a high throughput format, e.g., in a 96- well plate format.
  • RNA molecule corresponding to the third nucleic acid molecule may be used to direct in vitro translation to produce a polypeptide encoded by a sequence of interest present on the first nucleic acid molecule.
  • Any suitable in vitro translation protocol may be employed to translate the RNA into protein.
  • One suitable method is a bilayer method ( Figure 8). For example, an upper layer (e.g., of about 125 ⁇ l volume) containing a suitable substrate mixture, which may include, for example, creatin phosphate, GTP, ATP, and/or amino acids) may be added to a lower layer (e.g., of about 25 ⁇ l volume) comprising an extract suitable for in vitro translation and RNA corresponding to the sequence of interest encoding a polypeptide.
  • a suitable substrate mixture which may include, for example, creatin phosphate, GTP, ATP, and/or amino acids
  • RNA may be purified from the in vitro transcription reaction, for example, by ethanol precipitation, prior to being added to the extract. Typically, approximately 3 ⁇ l out of a 20 ⁇ l volume in vitro transcription reaction is added. The amount of RNA added can be varied as is known to those skilled in the art.
  • the layers may be incubated at a suitable temperature, for example, at 26 °C, for a suitable period of time, for example, 24 hours.
  • the transcription and translation reactions may be coupled, i.e., a PCR product may be added to a mixture containing a suitable RNA polymerase and the requisite materials for translation (e.g., a cell extract and suitable substrate mixture).
  • the reactions of methods of the invention may be performed sequentially, for example, in the same reaction vessel (e.g., micro fuge tube, well of a microtiter plate, etc.).
  • a recombination reaction may be conducted in a small volume (e.g., 5 ⁇ l).
  • the reaction may be stopped, for example, by heating to inactivate the recombination proteins, and then the material for an amplification reaction may be added directly to the recombination reaction.
  • buffers, primers, nucleotides, and a DNA polymerase may be added up to a second volume (e.g., 25 ⁇ l).
  • the amplification reaction may be conducted and them materials for an in vitro transcription reaction may be added (e.g., RNA polymerase and ribonucleotides) up to a third volume (e.g., 50 ⁇ l).
  • a materials for an in vitro translation reaction e.g., a cell extract and a substrate mixture
  • a fourth volume e.g., a cell extract and a substrate mixture
  • the products of the translation reaction may be analyzed and/or recovered using any suitable technology, for example, by SDS-PAGE, column chromatography, etc.
  • the translation reaction mixture may be clarified by centritugation. The mixture may be centrifuged at 14,500 rpm (19,000xg) for 20 min at 4 °C and the supernatant may be recovered.
  • the samples were applied to an SDS-polyacrylamide gel and separated using standard techniques.
  • the proteins were transferred to a membrane and analyzed using antibodies to a 6-histidine tag encoded by the second nucleic acid molecule (i.e., the destination vector pEU3'His in Figure 7).
  • the primary antibody was an anti-6-His antibody (His-probe (G-18), Santa Cruz Biotechnology, Inc., Santa Cruz, CA), used at 1/1000 dilution in PBST-3% BSA (phosphate buffered saline containing Tween-20 at 0.1% (v/v))
  • the secondary antibody was an anti-rabbit Ig, HRP-Linked Whole Ab, used at 1/2000 dilution in PBST-10% milk (Amersham Biosciences Anti-rabbit Ig, HRP-linked whole antibody (Donkey)cat. no. NA934). Detection was performed the detection reaction with ECL-Plus (Amersham Biosciences ECL PLUS Western blotting detection reagent, cat. no. RPN2132), and detected with BIO-RAD Fluor-S MAX according to the manufacturers instructions.
  • An additional benefit provided by the present invention is the reduction in the time required to produce a polypeptide from a sequence of interest. Using the above-described methods, a polypeptide may be produced much more rapidly than was previously possible. As shown in Figure 6, the present methods can substantially reduce the time required to produce proteins.
  • the above-described methods are particularly well suited to the parallel processing of a large number of sequences of interest, i.e., high throughput processing.

Abstract

La présente invention concerne des matériels et des méthodes de transcription et de traduction in vitro d'une séquence d'acide nucléique à étudier. Les méthodes peuvent consister en une réaction de recombinaison entre un acide nucléique comprenant une séquence à étudier et une molécule d'acide nucléique supplémentaire comprenant un ou plusieurs sites de recombinaison. L'invention concerne également des trousses de mise en oeuvre des méthodes de l'invention. L'invention concerne en outre des méthodes de mise en oeuvre d'une entreprise de fourniture des molécules d'acide nucléique et des polypeptides produits selon les méthodes de l'invention.
PCT/US2003/018219 2003-05-15 2003-06-12 Compositions et methodes de recombinaison et d'expression specifiques d'un site WO2004104194A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5888732A (en) * 1995-06-07 1999-03-30 Life Technologies, Inc. Recombinational cloning using engineered recombination sites
US6277608B1 (en) * 1997-10-24 2001-08-21 Invitrogen Corporation Recombinational cloning using nucleic acids having recombination sites

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5888732A (en) * 1995-06-07 1999-03-30 Life Technologies, Inc. Recombinational cloning using engineered recombination sites
US6277608B1 (en) * 1997-10-24 2001-08-21 Invitrogen Corporation Recombinational cloning using nucleic acids having recombination sites

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