EP1165768A1 - PROCEDES D'OBTENTION DE SEQUENCES D'ACIDE NUCLEIQUE PLEINE LONGUEUR A L'AIDE DE $i(E. COLI) TOPOISOMERASE III ET SES HOMOLOGUES - Google Patents

PROCEDES D'OBTENTION DE SEQUENCES D'ACIDE NUCLEIQUE PLEINE LONGUEUR A L'AIDE DE $i(E. COLI) TOPOISOMERASE III ET SES HOMOLOGUES

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Publication number
EP1165768A1
EP1165768A1 EP00912216A EP00912216A EP1165768A1 EP 1165768 A1 EP1165768 A1 EP 1165768A1 EP 00912216 A EP00912216 A EP 00912216A EP 00912216 A EP00912216 A EP 00912216A EP 1165768 A1 EP1165768 A1 EP 1165768A1
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European Patent Office
Prior art keywords
mrna
cdna
expression vector
sequence
tag
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EP00912216A
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German (de)
English (en)
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John A. Heyman
John D. Comiskey
Robert Marcil
Dorothy Phelan
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Life Technologies Corp
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Invitrogen Corp
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Publication of EP1165768A1 publication Critical patent/EP1165768A1/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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/90—Isomerases (5.)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4705—Regulators; Modulating activity stimulating, promoting or activating activity
    • 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
    • C12N15/1096—Processes for the isolation, preparation or purification of DNA or RNA cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the invention disclosed herein relates to the fields of molecular biology and genomics and methods useful therein. More specifically, the invention relates to methods useful for the cloning and characterization of nucleic acid sequences encoding a full-length protein.
  • genomic D ⁇ N is simply isolated, digested into fragments of a manageable size and sequenced using an automated sequencing system. Computerized searches of the resulting sequence data allow scientists to identify particular stretches of sequence that appear to encode a protein. This technique has proved useful when working with organisms having a relatively small genome.
  • the genomes of complex organisms, such as humans appear to contain a great deal of D ⁇ A sequence that does not encode proteins (introns and the like), thus blanket sequencing approaches may be a relatively inefficient means for identifying previously unknown, potentially useful proteins.
  • the relatively non-abundant messenger RNA is isolated from a population of cells by contacting total isolated RNA with a poly-T sequence bound to a solid support under conditions where the 3' poly-A tail of the mRNA will bind to the poly-T sequence. Unbound molecules are washed away and the mRNA released with a low salt wash.
  • cDNA is produced from the mRNA by 1) using reverse transcriptase to synthesize first strand DNA, 2) hydrolyzing the mRNA template by exposure to base, then 3) synthesizing second strand DNA with DNA polymerase. This results in a blunt-ended double-stranded sequence, which can then either be inserted into an expression vector directly or first tagged with a short linker nucleic acid having a restriction endonuclease recognition sequence contained therein.
  • the present invention comprises a method of obtaining full-length coding sequences by, in part, employing the attachment of a non-native tag to a single- stranded cDNA product.
  • the invention method comprises isolating full- length mRNA, using the isolated mRNA to synthesize first strand cDNA, attaching a non-native tag to the cDNA and using the tagged first strand to synthesize second strand cDNA.
  • the final cDNA can additionally be amplified, using the polymerase chain reaction for example, and inserted into an expression vector.
  • a further aspect of the invention comprises isolated full-length coding cDNA prepared using the method of the invention, as well as nucleic acid constructs containing the full-length coding cDNA.
  • the invention method is efficient and robust and provides cDNA products that can be of great benefit to those wishing to obtain and study novel proteins and/or use said proteins as a means to develop new and useful substances.
  • Figure 1 is a schematic representation showing attachment of a non-native tag to single-stranded cDNA using E. coli topoisomerase III.
  • the invention methods comprise a series of steps: isolating full-length mRNA, synthesizing first strand cDNA using the mRNA isolated in the first step as a template, attaching a non-native tag to the 3 '-end of the first strand cDNA and synthesizing second strand cDNA using the tagged first strand cDNA as a template.
  • the invention method is unique in that it employs a novel method for the attachment of a non-native tag to a single-stranded DNA, in particular a tag comprising a sequence of nucleic acids having additional utility, such as, for example, providing for convenient cloning activities.
  • coding sequence refers to the nucleic acids encoding the amino acid sequence of a protein
  • a “gene sequence” refers to the entire nucleic acid sequence that is necessary for the synthesis of a functional polypeptide.
  • a gene sequence could include, for example, a promoter in addition to a coding sequence.
  • cDNA is used to refer to DNA that is complementary to mRNA, that is, unlike genomic DNA, it lacks introns.
  • the mRNA used in the method of the invention is typically derived from total RNA isolated from a population of cells. Techniques for isolation of total RNA are well known in the art. Exemplary techniques are described in Ausubel, et al, Short Protocols in Molecular Biology, 3rd ed. 1995, which is incorporated by reference herein in its entirety.
  • Total RNA is a mixed population of transfer RNA (tRNA) and mRNA from which the mRNA must be separated prior to initiating cDNA synthesis.
  • tRNA transfer RNA
  • the most widely used technique exploits the poly- A tail found on the 5 '-end of mRNA, as described above. This technique results in a population of mRNA sequences that contains a mixture of full-length and partial rnRNAs, which then produces a population of cDNAs that will be a mixture of both full-length coding sequences and partial sequences.
  • a more preferred method useful with eukaryotic cells isolates only full-length mRNA sequences utilizing the 5 '-cap structure found on such sequences.
  • rnRNAs are modified at their 5 '-ends with a cap structure consisting of a 7-methylguanosine in a 5'-to-5' triphosphate linkage to the first transcribed nucleotide of the mRNA. This structure is added early during RNA transcription and is required as part of RNA biogenesis (see Sonenberg, N. Prog. Nucleic Acid Res. 35:173-207, 1988 for a review).
  • the cap structure can be employed to isolate full-length mRNA from other RNAs by several methods.
  • the cap structure of RNA total or mRNA previously isolated by oligo-dT techniques as described above
  • an affinity purification tag such as biotin, chitin binding domain, glutathione-S- transferase, and the like
  • the affinity tagged capped mRNA can then be isolated from degraded mRNA or RNAs with poly-A tails that are not full-length.
  • the affinity tagged mRNA is separated from untagged RNA using affinity purification, for example by contacting the tagged mRNA with an affinity purification material such as a solid support complexed with streptavidin, avidin, chitin, glutathione, and the like.
  • an affinity purification material such as a solid support complexed with streptavidin, avidin, chitin, glutathione, and the like.
  • Suitable solid supports include various column chromatography gels, such as sepharose, agarose, cellulose, and the like, and magnetic beads. Chromatography gels can be additionally modified with substances such as nickel.
  • unmodified capped mRNA can be separated from RNA species lacking a cap by several methods.
  • the capped mRNA can be contacted with a solid support complexed to, for example, phenylboronic acid or dihydroxylborate (see Theus and Liarakos, Biotechniques 9(5):610-612, 1990).
  • the boronate ligand binds specifically and reversibly to vicinal diols such as the 2',3'-cis- diol of 7-methylguanosine ribose.
  • the complex is disrupted by acidic pH and a chelating agent such as EDTA, thereby releasing the mRNA from the affinity purification material.
  • Unmodified capped rnRNAs may also be isolated using one or more cap- binding proteins bound to a solid support. Cap-binding proteins specifically recognize and bind to 7-methylguanosine. Several cap-binding proteins have been identified and isolated including eIF4E, eIF4A, eIF4G, eIF4B, and eIF4F, which exist in isomeric form in some species (see Haghighat and Sonenberg, JBC 272:21677- 21680, 1997; van Heerden and Browning, J Biol Chem 269:17454-17457, 1994), and nCBP (see Ruud, et al, JBC 273(17):10325-10330, 1998).
  • eIF4F is a multi-subunit complex composed of eIF4E, eIF4A and eIF4G.
  • a preferred method of isolating capped mRNA comprises using a eIF4E/eIF4G or nCBP/eIF4G complex bound to a solid support.
  • Such complexes can be comprised of isolated, complexed individual proteins, or alternatively, a fusion protein comprising the cap- binding portions of the relevant cap-binding proteins.
  • Complexes suitable for use in the present invention can be produced in a variety of ways.
  • Each of the components of the complex generally two, can be isolated separately from a natural or recombinant source then either sequentially immobilized on a solid support or first mixed and allowed to form complexes in solution, which are subsequently bound to a solid support.
  • a preferred method of forming complexes is to recombinantly produce them in the same cell line, so that complexes are formed within the cellular environment, purified, then bound to a solid support.
  • Affinity purification tags as described below, can be added to the recombinant proteins to aid in purification.
  • Fusion proteins that would be suitable for use in the practice of the present invention include a fusion between the C-terminus of full-length eI4E and the N-terminus of full-length eI4G, with or without the addition of an affinity purification tag, or the N- terminus of full-length eI4E fused to the C-terminus of full-length eI4G, with or without an affinity purification tag.
  • Any cell type can serve as a source for mRNA to be used in practicing the method of the invention including both eukaryotic cells and prokaryotic cells, although only eukaryotic cells (such as plant, animal or insect cells) can be used as a source for mRNA isolated by the cap-binding procedure described above.
  • eukaryotic cells such as plant, animal or insect cells
  • Suitable animal cells include mammalian cells (human, rodent, non-human primate, goat, sheep, cow, and the like) and insect cells (moth, Drosophila, and the like). Cells in any stage of differentiation are suitable as a source for mRNA, as are cells in any particular activation state. Methods of extracting mRNA from different cell types are well known in the art (see, for example, Ausubel, et al, supra).
  • the isolated mRNA (either eukaryotic or prokaryotic) is used as a template for the synthesis of the first, or anti-sense, strand of the cDNA.
  • Methods of first strand cDNA synthesis are well known in the art.
  • a poly(dT)-containing oligonucleotide is used as a primer for synthesis by a reverse transcriptase enzyme.
  • First strand synthesis techniques are described in detail in Ausubel, supra, and in the Examples below.
  • the mRNA/cDNA hybrids are treated with a substance that will degrade the mRNA, such as, for example, NaOH, yielding single-stranded first strand cDNA.
  • a substance that will degrade the mRNA such as, for example, NaOH
  • the mRNA/cDNA hybrid may first be treated with a substance that degrades single- stranded mRNA, such as, for example, RNAse.
  • any of the 5'- capped ends of any mRNA cDNA hybrids wherein the cDNA is not full-length will be removed.
  • RNAse treatment After RNAse treatment, the treated hybrids are subjected to a cap protein-based affinity purification step as described above, removing any hybrids in which the first strand cDNA is not full-length. The isolated full-length RNAse- treated hybrids are then treated to degrade the mRNA, as described above.
  • a non-native tag sequence is next attached to the first strand cDNA.
  • Scheele US Patent 5,162,209, issued 11/10/92 describes a method wherein a homopolymeric oligonucleotide tag is added to the 3' end of first strand cDNA using an enzyme such as terminal deoxyribonucleotidyl transferase, polyA polymerase, and the like.
  • the resulting DNA either possesses a persistent homopolymeric oligonucleotide tag with no utility or, using the method described by Scheele, the tag is removed and the resulting cDNA is left with blunt ends, which reduces later cloning efficiency.
  • T4 RNA ligase An alternative method of adding a non-native tag sequence to single-stranded DNA uses T4 RNA ligase (see Tessier, et al., Anal Biochem 158:174-178, 1986; McCoy and Gumport, Biochemistry 19:635-642, 1980). This enzyme is, however, relatively slow and inefficient and does not work efficiently with any sequence (see Harada and Orgel, Proc. Natl. Acad. Sci. USA 90:1576-1579, 1993).
  • the non-native tags preferably employed in the invention method are nucleic acids which have some utility, such as comprising an enzyme recognition sequence that can be employed in subsequent cloning and/or insertion into a nucleic acid construct.
  • a non-native tag sequence is attached to the 3 '-end of the first strand cDNA by a type I topoisomerase such as E. coli topoisomerase III, yeast topoisomerase III, and the like (see DiGate and Marrrians, JBC 264:17924-17930, 1989; Kim and Wang, JBC 267:17178-17185, 1992).
  • a type I topoisomerase such as E. coli topoisomerase III, yeast topoisomerase III, and the like (see DiGate and Marrrians, JBC 264:17924-17930, 1989; Kim and Wang, JBC 267:17178-17185, 1992).
  • Type I topoisomerases recognize and bind to specific recognition sequences in single-stranded DNA, catalyze a strand break and strand passage event, then reseal the break.
  • An enzyme-DNA adduct is formed at the point of DNA cleavage, with the enzyme covalently bound
  • topo III E. coli topoisomerase III
  • Topo III is a type I topoisomerase which recognizes, binds to and cleaves the single-stranded sequence 5'-GCAACTT-3' (S ⁇ Q. ID NO:l)(Zhang, et al, J. Biol. Chem. 270(40):23700-23705, 1995).
  • S ⁇ Q. ID NO:l the single-stranded sequence
  • a homolog, the tra ⁇ protein of plasmid RP4 has been described by Li, et al., J. Biol. Chem 272:19582-19587, 1997.
  • a DNA-protein adduct is formed with the enzyme covalently binding to the 5'- thymidine residue, with cleavage occurring between the two thymidines.
  • the non-native tag can be prepared by incubating a single-stranded nucleic acid molecule with topo III under conditions suitable for the enzyme's activity (discussed in greater detail in the Examples below).
  • the nucleic acid will contain the topo III recognition sequence and generally a second sequence that may be used for priming second strand synthesis and/or in subsequent cloning activities, such as, for example, a recognition sequence for a restriction endonuclease or a recombinase which recognizes and acts on double-stranded DNA sequences such as Cre, Flp or vaccinia topoisomerase.
  • a recognition sequence for a restriction endonuclease or a recombinase which recognizes and acts on double-stranded DNA sequences such as Cre, Flp or vaccinia topoisomerase.
  • the enzyme will bind to and cleave the nucleic acid molecule at the enzyme's recognition site and will, in the case of E. coli topoisomerase III, become covalently linked to the DNA 3' to the cleavage site via an enzyme-bridged phosphotyrosine linkage.
  • This adduct can be isolated from cleavage products by well known treatment methods, for example treatment with detergents to temporarily inactivate the enzyme, followed by purification of the complex by standard chromatography or electrophoresis techniques, both of which are well known in the art.
  • the tag- formation reaction can be performed while one end of the uncleaved molecule is immobilized and the clipped pieces removed by continual electrophoresis within an electric field.
  • the pre-cleaved nucleic acid molecule can be designed to contain a bridging phosphorothioate between the two thymidines of the GCAACTT cleavage/recognition sequence.
  • the clipped piece will contain a 3'-SH instead of a 3' -OH, preventing religation (see Burgin, et al, Nucleic Acids Res 23:2973-2979, 1995).
  • the DNA "tag” can be a nucleic acid vector, rather than a shorter nucleic acid molecule.
  • Suitable vectors can be prepared as follows.
  • a nucleic acid molecule suitable for attachment to a vector can be created by synthesizing complementary single strands of DNA which when annealed leave a single-stranded overhang containing a topoisomerase III recognition/cleavage sequence.
  • the double stranded end of the attachment molecule can either be blunt or contain a restriction endonuclease cutting sequence.
  • a vector (as described below) can be linearized using a restriction endonuclease in such a way that the 5' end will match that of the attachment molecule.
  • the attachment molecule is similarly treated, then ligated to the vector, forming a vector with a single stranded overhang containing the topoisomerase III recognition site.
  • the modified vector is treated as described above to create an enzyme:vector adduct.
  • topo II tag adduct is incubated with the isolated first strand cDNA under conditions such that the enzyme will catalyze the joining reaction between the 3'-OH of the cDNA and the 5' end of the tag. Suitable conditions are discussed in greater detail in the Examples below.
  • the tagged cDNA is used as a template to synthesize second strand cDNA using the tagged first strand as a template.
  • Methods of producing second strand cDNA are well known in the art (see, Ausubel, supra).
  • One particularly useful method is the polymerase chain reaction (PCR), a technique well known in the art.
  • PCR polymerase chain reaction
  • the use of PCR has the advantage of amplifying the number of copies of any particular DNA sequence in addition to the creation of double-stranded DNAs.
  • Nucleic acids complementary to the non-native tag and either a poly-dT or a nucleic acid complementary to the 5' end of the DNA can be used as primers for strand extension.
  • DNA polymerase can be used to fill in the single stranded portions, creating blunt ends which can be ligated with T4 DNA ligase to create circular and transformable DNA according to techniques well known in the art.
  • the procedure described above will efficiently produce full-length protein-encoding nucleic acids.
  • the proteins encoded thereby can then be produced by inserting the newly cloned nucleic acid into an expression vector, which is subsequently transfected into a host cell for protein expression.
  • the amplified double-stranded DNAs can be isolated from the other components of the amplification reaction mixture prior to insertion into an expression vector.
  • This purification can be accomplished using a variety of methodologies such as column chromatography, gel electrophoresis, and the like.
  • One method of purification utilizes low-melt agarose gel electrophoresis. The reaction mixture is separated and visualized by ethidium bromide staining. DNA bands that represent a majority of the amplification products are cut away from the rest of the gel and placed into appropriate corresponding wells of a 96-well microtiter plate. These plugs are subsequently melted and the DNA contained therein utilized as cloning inserts.
  • the use of gel electrophoresis has the advantage that the practitioner can simultaneously purify the desired nucleic acid and verify that the sequence is of a reasonable size, i.e., probably represents the entire desired coding sequence.
  • the purified coding sequence can then be inserted into an expression vector.
  • expression vectors are suitable for use in the method of the invention, both for prokaryotic expression and eukaryotic expression.
  • the expression vector will have one or more of the following features: a promoter-enhancer, a selection marker, an origin of replication, an affinity purification tag, an inducible element, an epitope-tag, and the like.
  • Promoter-enhancers are DNA sequences to which RNA polymerase binds and initiates transcription. The promoter determines the polarity of the transcript by specifying which strand will be transcribed.
  • Bacterial promoters consist of -35 and - 10 (relative to the transcriptional start) consensus sequences which are bound by a specific sigma factor and RNA polymerase. Eukaryotic promoters are more complex. Most promoters utilized in expression vectors are transcribed by RNA polymerase II.
  • General transcription factors (GTFs) first bind specific sequences near the start and then recruit the binding of RNA polymerase II.
  • GTFs General transcription factors
  • Viral promoters serve the same function as bacterial or eukaryotic promoters and either provide a specific RNA polymerase in trans (bacteriophage T7) or recruit cellular factors and RNA polymerase (SV40, RSV, CMV). Viral promoters are preferred as they are generally particularly strong promoters. Promoters may be, furthermore, either constitutive or, more preferably, regulatable (i.e., inducible or derepressible). Inducible elements are DNA sequence elements which act in conjunction with promoters and bind either repressors (e.g. lacO/LAC Iq repressor system in E. coli) or inducers (e.g. gall/GAL4 inducer system in yeast). In either case, transcription is virtually "shut off' until the promoter is derepressed or induced, at which point transcription is "turned-on".
  • repressors e.g. lacO/LAC Iq repressor system in E. coli
  • inducers e.g.
  • constitutive promoters include the int promoter of bacteriophage ⁇ , the bla promoter of the ⁇ -lactamase gene sequence of pBR322, the CAT promoter of the chloramphenicol acetyl transferase gene sequence of pPR325, and the like.
  • inducible prokaryotic promoters include the major right and left promoters of bacteriophage (P L and P R ), the trp, reca, lacZ, Lad, AraC and gal promoters of E. coli, the ⁇ -amylase (Ulmanen Ett at., J. Bacteriol. 162:176-182, 1985) and the sigma-28-specific promoters ofB.
  • subtilis (Gilman et al., Gene sequence 32:11-20, 1984), the promoters of the bacteriophages of Bacillus (Gryczan, In: The Molecular Biology of the Bacilli, Academic Press, Inc., NY (1982)), Streptomyces promoters (Ward et at., Mol. Gen. Genet. 203:468-478, 1986), and the like.
  • Exemplary prokaryotic promoters are reviewed by Glick (J. Ind. Microtiot. 1 :277- 282, 1987); Cenatiempo (Biochimie 68:505-516, 1986); and Gottesman (Ann. Rev. Genet. 18:415-442, 1984).
  • Preferred eukaryotic promoters include, for example, the promoter of the mouse metallothionein I gene sequence (Hamer et al., J. Mol. Appl. Gen. 1:273-288, 1982); the TK promoter of Herpes virus (McKnight, Cell 31:355-365, 1982); the SV40 early promoter (Benoist et al., Nature (London) 290:304-310, 1981); the yeast gal4 gene sequence promoter (Johnston et al., Proc. Natl. Acad. Sci. (USA) 79:6971-6975, 1982); Silver et al., Proc. Natl. Acad. Sci. (USA) 81:5951-5955,
  • the CMV promoter the CMV promoter, the EF-1 promoter, ecdysone-responsive promoter, and the like.
  • Selection markers are valuable elements in expression vectors as they provide a means to allow only those cells which contain a vector to grow. Such markers are of two types: drug resistance and auxotrophic.
  • a drug resistance marker enables cells to detoxify an exogenously added drug that would otherwise kill the cell.
  • Auxotrophic markers allow cells to synthesize an essential component (usually an amino acid) while grown in media which lacks that essential component.
  • selectable markers include those which encode resistance to antibiotics such as ampicillin, tetracycline, kannamycin, streptomycin, hygromycin, neomycin, ZeocinTM, and the like.
  • Selectable auxotrophic genes include, for example, hisD, which allows growth in histidine free media in the presence of histidinol.
  • a preferred selectable marker for use in yeast expression systems is URN3.
  • Laboratory yeast strains carrying mutations in the gene which encodes orotidine-5'- phosphate decarboxylase, an enzyme essential for uracil biosynthesis, are unable to grow in the absence of exogenous uracil.
  • a copy of the wild-type gene (ura4+ in S. pombe and URN3 in S. cerevisiae) will complement this defect in trans.
  • a further element useful in an expression vector is an origin of replication.
  • Replication origins are unique D ⁇ A segments that contain multiple short repeated sequences that are recognized by multimeric origin-binding proteins and which play a key role in assembling D ⁇ A replication enzymes at the origin site.
  • Suitable origins of replication for use in expression vectors employed herein include E. coli oriC, 2 ⁇ and ARS (both useful in yeast systems), Col ⁇ l, sfl, SV40 (useful in mammalian systems), and the like.
  • Additional elements that can be included in an expression vector employed in the invention method are sequences encoding affinity purification tags or epitope tags.
  • Affinity purification tags are generally short peptides that can interact with a binding partner immobilized on a solid support.
  • Synthetic D ⁇ As encoding multiple consecutive single amino acids, such as histidine, when fused to the expressed protein, may be used for one-step purification of the recombinant protein by high affinity binding to a resin column, such as nickel sepharose.
  • An endopeptidase recognition sequence is engineered between the polyamino acid tag and the protein of interest to allow subsequent removal of the leader peptide by digestion with ⁇ nterokinase.
  • Sequences encoding peptides such as the chitin binding domain (which binds to chitin), glutathione-S-transferase (which binds to glutathione), Thio-Patch (LaVallie, et al. Bio/Technology 11:187-193, 1993) (which binds to metal-chelating resins), and the like can also be used for facilitating purification of the protein of interest.
  • the affinity purification tag can be separated from the protein of interest by methods well known in the art, including the use of inteins (protein self-splicing elements, Chong, et al, Gene 192:271-281, 1997).
  • Epitope tags are short peptides that are recognized by epitope specific antibodies.
  • a fusion protein comprising a recombinant protein and an epitope tag can be simply and easily purified using an antibody bound to a chromatography resin.
  • the presence of the epitope tag furthermore allows the recombinant protein to be detected in subsequent assays, such as Western blots, without having to produce an antibody specific for the recombinant protein itself.
  • Examples of commonly used epitope tags include V5, glutathione-S-transferase (GST), hemaglutinin (HA), the peptide Phe-His-His-Thr-Thr (SEQ ID NO:2), chitin binding domain, and the like.
  • a further useful element in an expression vector is a multiple cloning site or polylinker.
  • Synthetic DNA encoding a series of restriction endonuclease recognition sites is inserted into a plasmid vector downstream of the promoter element. These sites are engineered for convenient cloning of DNA into the vector at a specific position.
  • Suitable prokaryotic vectors include plasmids such as those capable of replication in E. coli (for example, pBR322, ColEl, pSClOl, PACYC 184, itVX, pRSET, pBAD (Invitrogen, Carlsbad, CA), and the like).
  • plasmids are disclosed by Sambrook (cf. "Molecular Cloning: A Laboratory Manual", second edition, edited by Sambrook, Fritsch, & Maniatis, Cold Spring Harbor Laboratory, (1989) the relevant sections of which are incorporated by reference herein).
  • Bacillus plasmids include pC194, pC221, pT127, and the like, and are disclosed by Gryczan (In: The Molecular Biology of the Bacilli, Academic Press, NY (1982), pp. 307-329).
  • Suitable Streptomyces plasmids include plJlOl (Kendall et al., J. Bacteriol. 169:4177- 4183,1987), and Streptomyces bacteriophages such as ⁇ C31 (Chater et al., In: Sixth International Symposium on Actinomycetales Biology, Akademiai Kaido, Budapest, Hungary (1986), pp. 45-54).
  • Pseudomonas plasmids are reviewed by John et al. (Rev. Infect. Dis. 8:693-704, 1986), and Izaki (Jpn. J. Bacteriol. 33:729-742, 1978).
  • Suitable eukaryotic plasmids include, for example, BPV, vaccinia, SV40, 2- micron circle, pCDNA3.1, pCDNA3.1/GS, pYES2/GS, pMT, pIND, pIND(Spl), pVgRXR (Invitrogen), and the like, or their derivatives.
  • Such plasmids are well known in the art (Botstein et al., Miami Wntr. Symp. 19:265-274, 1982; Broach, In: "The Molecular Biology of the Yeast Saccharomyces: Life Cycle and Inheritance", Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, p.
  • the coding sequences can be inserted into an expression vector using any of a variety of techniques.
  • the non-native tag added to the cDNA contains a restriction endonuclease recognition sequence
  • the coding sequence can be ligated into a compatible sequence existing in the desired expression vector using standard techniques.
  • a particularly preferred method is the use of the vaccinia topoisomerase cloning system described in US Patent No. 5,766,891, issued June 16, 1998 to S. Shurnan and available as a cloning kit from Invitrogen, Corp., Carlsbad, CA.
  • Prokaryotic hosts are, generally, very efficient and convenient for the production of recombinant proteins and are, therefore, one type of preferred expression system. Prokaryotes most frequently are represented by various strains of E. coli. However, other organisms may also be used, including other bacterial strains. The prokaryotic host selected for use herein must be compatible with the replicon and control sequences in the expression plasmid. Recognized prokaryotic hosts include bacteria such as E. coli and those from genera such as Bacillus, Streptomyces, Pseudomonas, Salmonella, Serratia, and the like. However, under such conditions, the polypeptide will not be glycosylated.
  • Suitable hosts may often include eukaryotic cells.
  • Preferred eukaryotic hosts include, for example, yeast, fungi, insect cells, and mammalian cells either in vivo, or in tissue culture.
  • Mammalian cells which may be useful as hosts include HeLa cells, cells of fibroblast origin such as VERO, 3T3 or CHOK1, HEK 293 cells or cells of lymphoid origin (such as 32D cells), and their derivatives.
  • Preferred mammalian host cells include non-adherent cells such as CHO, 32D, and the like.
  • Preferred yeast host cells include S. pombe, S. cerevisiae (such as INVScl), and the like.
  • plant cells are also available as hosts, and control sequences compatible with plant cells are available, such as the cauliflower mosaic virus 35S and 19S, nopaline synthase promoter and polyadenylation signal sequences, and the like.
  • Another preferred host is an insect cell, for example the Drosophila larvae. Using insect cells as hosts, the Drosophila alcohol dehydrogenase promoter can be used, (Rubin, Science 240:1453-1459, 1988).
  • baculovirus vectors can be engineered to express large amounts of peptide encoded by a desired gene sequence in insect cells (Jasny, Science 238:1653, 1987); Miller et al., In: Genetic Engineering (1986), Setlow, J.K., et al., eds., Plenum, Vol. 8, pp. 277-297).
  • the present invention also features the purified, isolated or enriched versions of the expressed coding sequence products produced by the methods described above.
  • Full-length mRNA is isolated from a population of cells by first isolating total
  • RNA using a commercially available kit, RNA Isolation Reagent (RNAwiz, Ambion) according to the manufacturer's instructions.
  • the 560 bp encoded wheat germ eIF-4E protein (the p86 subunit, Van Heerden and Browning, supra) is cloned into a vector such as pCR4 (Invitrogen), according to the manufacturer's instructions, to produce a Thio-Patch fusion protein.
  • pCR4 Invitrogen
  • a chitin binding domain encoding sequence is also cloned upstream of the Thio-Patch site (pCR4), so that the resulting expressed eIF-4E protein contains both a chitin binding domain and Thio-Patch at the N- terminus.
  • a stop codon is introduced at the end of the insert so that the coding sequence will not read-through to the V5 epitope contained on the vector.
  • Lysates are prepared by resuspending a liter of pelleted cells in Buffer A lysis solution (25 - 30 ml, Buffer A (10 mM tris, pH 7.5, 0.2 mM ⁇ DTA, 1 mM DTT, 10% glycerol, 100 mM KC1) plus 0.5% Triton X- 100, 1 mM PMSF (phenylmethylsulfonyl fluoride)), repeated sonication, then centrifugation at 12,000 rpm for 15 minutes. The cleared lysate is removed from the pelleted cell debris and 1 mM PMSF added.
  • Buffer A 10 mM tris, pH 7.5, 0.2 mM ⁇ DTA, 1 mM DTT, 10% glycerol, 100 mM KC1
  • Triton X- 100, 1 mM PMSF (phenylmethylsulfonyl fluoride) repeated sonication, then centrifugation at 12,000 rpm
  • the protein is purified using m 7 GTP Sepharose (Pharmacia).
  • the resin is equilibrated with Buffer A, then added to the cleared lysate and stirred slowly overnight at 4°C.
  • the resin is pelleted by centrifugation and the liquid removed.
  • the resin is resuspended in Buffer A, washed by agitation at room temperature for 30 minutes, then re-pelleted. After removal of the liquid, the washed resin is resuspended in Buffer A and loaded into an empty column, allowing the Buffer A to drain through.
  • the column is continuously washed with Buffer A until no protein is detected in the flow-through.
  • the cap binding protein is eluted with 70 ⁇ M m 7 GTP in Buffer A.
  • RNA is added to the resin in a binding buffer containing 10 mM KHPO 4 (pH8), 100 mM KC1, 5% glycerol, 2mM EDTA, and 6 mM dithiothreitol (DTT) (added just before using) and supplemented with 1.3% polyvinyl alcohol (PVA).
  • PVA polyvinyl alcohol
  • RNA pellet After the final wash is removed, 600 ⁇ l of lOmM Tris (pH8) is added to the resin.
  • the bound mRNA is eluted using an equal volume of phenol/chloroform, pH8. Following elution, the mRNA is precipitated with 0.4M NaOAc and an equal volume of isopropanol at -70°C for at least 30 minutes, then spun down in a microfuge to collect the RNA pellet. 20 ⁇ g of mussel glycogen is added as a carrier to aid in precipitation. The mRNA pellet is resuspended in 20 ⁇ RNAse-free water.
  • the isolated mRNA is converted to first-strand cDNA using a cDNA Cycle® Kit (Invitrogen) using the oligo dT primer provided and the protocols suggested by the manufacturer.
  • the RNA is hydrolyzed by exposure to 0.2 N NaOH at 37° C for 30 minutes.
  • the resulting single-stranded cDNA is tagged by the addition of previously prepared tag covalently bound to topoisomerase III (see below) in Tris pH8.0, lmM Mg acetate, O.l ⁇ g/ml BSA, NaCl for 5 minutes at 37°C.
  • Topoisomerase II DNA tags can be prepared by incubating a sequence such as, for example, 5'-NNNGCAACT*TCCCTATAGTGAGTCGTATTA-3' (SEQ ID NO:3) withE. coli topoisomerase III in 40 mM Hepes-KOH (pH 8.0 at 22°C), 0.1 mg/ml bovine serum albumin, and 1 mM magnesium acetate (pH 7.0) at 37°C for 3 - 5 minutes (where * marks the cleavage point). The reaction can be stopped by the addition of SDS or another mild detergent to a final concentration of 2%.
  • the covalent enzyme:DNA adduct is isolated using column chromatography, for example using a size exclusion resin and HPLC.
  • PCR can be performed using the cDNA Cycle Kit according to the manufacturer's instructions and primers corresponding to the 3' end of the tag sequence and oligo-dT.
  • the reaction cycles can be as follows: 2 minutes at 94°C, then 25 - 35 cycles (10 sec/cycle) at 94°C, 55°C and 72°C, followed by 5 minutes at 72°C.
  • the resulting amplified cDNA can be inserted into a plasmid vector such as pCR®2 or pCR®2-TopoTM (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions.

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Abstract

L'invention porte sur un procédé d'obtention d'un ADNc codant pleine longueur. Ce procédé consiste à isoler un ARNm pleine longueur afin de synthétiser un premier brin d'ADNc, à fixer une étiquette non indigène sur l'ADNc et à utiliser le premier brin étiqueté pour synthétiser le second brin d'ADNc. L'ADNc final peut être ensuite amplifié et inséré dans un vecteur d'expression. L'invention porte également sur des ADNc codant pleine longueur préparés selon ce procédé, ainsi que sur des produits de recombinaison d'acide nucléique contenant les ADNc codant pleine longueur.
EP00912216A 1999-03-19 2000-03-13 PROCEDES D'OBTENTION DE SEQUENCES D'ACIDE NUCLEIQUE PLEINE LONGUEUR A L'AIDE DE $i(E. COLI) TOPOISOMERASE III ET SES HOMOLOGUES Withdrawn EP1165768A1 (fr)

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US12512699P 1999-03-19 1999-03-19
US125126P 1999-03-19
PCT/US2000/006560 WO2000056878A1 (fr) 1999-03-19 2000-03-13 Procedes d'obtention de sequences d'acide nucleique pleine longueur a l'aide de e. coli topoisomerase iii et ses homologues

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US5766891A (en) 1994-12-19 1998-06-16 Sloan-Kettering Institute For Cancer Research Method for molecular cloning and polynucleotide synthesis using vaccinia DNA topoisomerase
EP0920526B1 (fr) 1997-06-12 2004-11-24 Sloan-Kettering Institute For Cancer Research Assemblage covalent de brins d'adn a des brins d'arn catalyse par une vaccinia topoisomerase
US6326175B1 (en) * 1999-07-13 2001-12-04 Incyte Pharmaceuticals, Inc. Methods and compositions for producing full length cDNA libraries
JP2003524422A (ja) 2000-02-25 2003-08-19 インヴィトロジェン コーポレーション トポイソメラーゼリンカー介在型増幅法
US7198924B2 (en) 2000-12-11 2007-04-03 Invitrogen Corporation Methods and compositions for synthesis of nucleic acid molecules using multiple recognition sites
AU8835601A (en) 2000-08-21 2002-03-04 Invitrogen Corp Methods and reagents for molecular cloning
US20040166499A1 (en) 2000-10-05 2004-08-26 Yoshihide Hayashizaki Oligonucleotide linkers comprising a variable cohesive portion and method for the praparation of polynucleotide libraries by using said linkers
US20060008817A1 (en) 2000-12-08 2006-01-12 Invitrogen Corporation Methods and compositions for generating recombinant nucleic acid molecules
WO2002061034A2 (fr) 2000-12-08 2002-08-08 Invitrogen Corporation Compositions et methodes permettant de produire rapidement des molecules d'acide nucleique recombinees
US8304189B2 (en) 2003-12-01 2012-11-06 Life Technologies Corporation Nucleic acid molecules containing recombination sites and methods of using the same

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CA2005252C (fr) * 1988-12-13 2001-07-17 Nahum Sonenberg Isolement de clones d'adn-c pleine longueur et de mrna chapeautes
JP3337748B2 (ja) * 1992-09-25 2002-10-21 財団法人神奈川科学技術アカデミー 完全長ｃＤＮＡの合成方法、その中間体の製造方法及び完全長ｃＤＮＡを含む組換えベクターの製造方法
US5766891A (en) * 1994-12-19 1998-06-16 Sloan-Kettering Institute For Cancer Research Method for molecular cloning and polynucleotide synthesis using vaccinia DNA topoisomerase
FR2733762B1 (fr) * 1995-05-02 1997-08-01 Genset Sa Methode de couplage specifique de la coiffe de l'extremite 5' d'un fragment d'arnm et preparation d'arnm et d'adnc complet
US5962271A (en) * 1996-01-03 1999-10-05 Cloutech Laboratories, Inc. Methods and compositions for generating full-length cDNA having arbitrary nucleotide sequence at the 3'-end
JP3441899B2 (ja) * 1996-11-01 2003-09-02 理化学研究所 完全長ｃＤＮＡライブラリーの作成方法
US6187544B1 (en) * 1997-06-04 2001-02-13 Smithkline Beecham Corporation Methods for rapid cloning for full length cDNAs using a pooling strategy
EP0920526B1 (fr) * 1997-06-12 2004-11-24 Sloan-Kettering Institute For Cancer Research Assemblage covalent de brins d'adn a des brins d'arn catalyse par une vaccinia topoisomerase

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