WO2003010176A2 - Complexes acide nucleique-proteine - Google Patents

Complexes acide nucleique-proteine Download PDF

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Publication number
WO2003010176A2
WO2003010176A2 PCT/GB2002/003351 GB0203351W WO03010176A2 WO 2003010176 A2 WO2003010176 A2 WO 2003010176A2 GB 0203351 W GB0203351 W GB 0203351W WO 03010176 A2 WO03010176 A2 WO 03010176A2
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protein
sequence
nucleic acid
dna
sapv
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PCT/GB2002/003351
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English (en)
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WO2003010176A3 (fr
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Mikhail Soloviev
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Oxford Glycosciences (Uk) Ltd
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Priority to AU2002317380A priority Critical patent/AU2002317380A1/en
Publication of WO2003010176A2 publication Critical patent/WO2003010176A2/fr
Publication of WO2003010176A3 publication Critical patent/WO2003010176A3/fr

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y15/00—Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
    • C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
    • C07K14/3156—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci from Streptococcus pneumoniae (Pneumococcus)
    • 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
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1062—Isolating an individual clone by screening libraries mRNA-Display, e.g. polypeptide and encoding template are connected covalently
    • 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
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1075—Isolating an individual clone by screening libraries by coupling phenotype to genotype, not provided for in other groups of this subclass

Definitions

  • the invention provides novel methods for the production of recombinant proteins or protein oligomers that self-assemble into complexes with nucleic acids in solution or, when the nucleic acid is presented, or is presentable as an array, that self-assemble to produce a protein array.
  • Protein microarrays are potentially powerful tools in proteomics.
  • a protein function array can consist of thousands of native proteins immobilized on a substrate ready for parallel testing of protein function.
  • protein-binding agents may be arrayed, permitting expression profiling at the protein level.
  • Different methods are known in the art for attaching proteins to solid supports but these can involve cross-linking and loss of protein function (see PCT Application No. WO 00/32823).
  • enough purified protein needs to be prepared in its native conformation, something that is difficult to ensure. Indeed, many proteins are not amenable to over-expression in, for example, E. coli or baculovirus systems. Therefore, proteins expressed using in vitro transcription/translation methods where correctly-folded protein can be produced, are more attractive.
  • the direct transfer of technologies used in nucleic acid-based screening approaches to protein-based screening approaches is not possible because of the fundamentally different nature of nucleic acids and proteins.
  • the advantages conferred by the use of the polymerase chain reaction (PCR) in nucleic acid-based approaches are irrelevant to protein-based approaches because that technology does not result in the amplification of protein, and there is no equivalent means for the amplification of proteomes, or even mixtures of proteins, in vitro.
  • the present invention provides a method for the production of self-assembling protein-nucleic acid complexes using transcription followed by translation or using coupled in vitro transcription/translation systems, thus providing an efficient cloning vehicle for the cost- effective production of quantities of proteins of interest.
  • the invention provides a method for the production of a protein-nucleic acid complex comprising:
  • the term 'protein' includes any molecule that comprises a sequence of naturally- occurring or non-naturally-occurring amino acid residues. Amino acid residues that are post- translationally modified are also included, as are polypeptides, oligopeptides and peptides.
  • 'Cognate sequence' or 'cognate biomolecular sequence' includes a sequence that is recognized by, or recognises a binding partner such that an interaction occurs.
  • a sequence may comprise nucleic acid, amino acid, or other biomolecular or molecular sequences.
  • binding or interactions for example but without limitation, can be specific or non-specific, covalent or non-covalent, ionic or non-ionic.
  • the complexes of the present invention are protein-DNA complexes but it is also apparent that the method of the invention also applies to the preparation of protein- RNA self-assembled complexes.
  • the following text will now refer to protein-DNA complexes by way of explanation but not of limitation.
  • 'Protein vectors' comprise (i) an assembly sequence that permits the binding of the protein vector to its encoding DNA and (ii) a target protein sequence, i.e. the protein sequence of interest.
  • the 'assembly sequence' includes that part of a protein vector which binds to a cognate biomolecular sequence or site, for the purpose of immobilizing said protein vector directly or indirectly to its encoding DNA in a sequence specific or non-specific manner.
  • an assembly sequence can consist of, or comprise a target sequence.
  • a protein-DNA complex is formed i.e. a 'self-assembled' protein-DNA complex.
  • the assembly sequence binds indirectly to its encoding DNA by binding to a cognate sequence, e.g. a label, associated with the encoding, for example but without limitation, by labelling the encoding DNA with an appropriate molecule or ligand comprising said cognate sequence using methods known in the art. Labelling of the encoding DNA can be performed during transcription or translation or alternatively can be performed before or afterwards.
  • the protein vector assembly sequence (which is a cognate sequence for the ligand used to label the DNA) binds to the label attached to its encoding DNA.
  • Such labelled DNA and corresponding protein vectors comprise, for example but without limitation, interactors such as biotin and avidin, streptavidin or related proteins, derivatives or protein fragments that bind biotin, where biotin is the DNA label and avidin, streptavidin etc., is the assembly sequence of the protein vector encoded by the labelled DNA.
  • sequence-specific pairs of interactions can be used to produce self-assembled protein-DNA complexes. Examples of these include, but are not limited to, providing a nucleic acid comprising code for a binding site of a nucleic acid-binding protein and providing a protein vector comprising the cognate nucleic acid-binding protein as the target protein. Proteins that bind to nucleic acids are known in the art (see for example US 5,270,163). The use of known transcription factors and their target DNA sequences is a preferred example of such sequence-specific interactions.
  • binding of the assembly sequence to its encoding DNA occurs during protein synthesis whilst the peptide chain of the protein vector is still attached to a ribosome, i.e. the nascent protein binds to its encoding DNA.
  • binding occurs upon the completion of protein synthesis after the protein vector has been released from the ribosome.
  • complexes can be assembled step-wise, by firstly producing the protein vector using an appropriate protein expression system followed by addition of DNA encoding said protein vector, resulting in the association of the protein vector and its encoding DNA via the assembly sequence and hence the formation of a protein-DNA complex.
  • the protein vector of the protein-DNA complex may also bind to a suitable substrate directly or indirectly, or to a suitable molecule or ligand comprising a cognate sequence which can be attached to a suitable substrate.
  • suitable substrate' refers to any suitable solid or semi-solid support such as, but not limited to, glass, silicon, bead of material or other suitable materials.
  • an assembly sequence can comprise a target protein sequence wherein the target protein sequence is present within the assembly sequence i.e. interrupting the assembly sequence.
  • the target protein sequence preferably does not substantially alter the folding, if any, of the assembly sequence. In effect the target protein is "displayed" within the assembly sequence.
  • the target protein sequence is not present within the assembly sequence but is present elsewhere within the protein vector, preferably C-terminal to the assembly sequence or alternatively, N- terminal to the assembly sequence.
  • the protein vector additionally comprises an affinity tag.
  • the affinity tag may be adapted to permit purification of the protein-DNA complex.
  • the affinity tag may also permit identification and/or quantitation of a target protein/protein interaction or the interaction of the protein-DNA complex with another compound of interest.
  • 'Compounds of interest' include biomolecules such as proteins, polypeptides, peptides, antibodies, antigens, nucleic acids and also other organic and non-organic molecules.
  • the term 'affinity tag' is well known in the art and includes a region of protein sequence that recognises or is recognised by a ligand or other molecule or biomolecule.
  • Biomolecules include proteins, nucleic acids and other molecules of biological origin.
  • the affinity tag supplies a means of isolating the protein-DNA complex from a solution or a mixture of protein-DNA complexes in solution
  • the protein-DNA complex may comprise at least two different affinity tags.
  • the affinity tag of the protein vector serves to immobilise the protein- DNA complex to a suitable substrate either directly or indirectly.
  • the protein-DNA complexes of the invention can be presented in a spatial format as an array.
  • the affinity tag is preferably present at the opposite end of the protein vector to the assembly sequence, e.g. the assembly sequence and the affinity tag are situated at, or near, opposite termini of the target protein sequence.
  • the affinity tag can, without limitation, be adjacent to the assembly sequence or separated from the assembly sequence by a linker (see below), which can be cleavable or non-cleavable.
  • the DNA component of the protein-DNA complex can be used as a unique tag for characterising protein/protein interactions and hence can be used for separation, purification, identification and/or quantitation of a protein-DNA complex protein interaction or the interaction of the protein-DNA complex of the invention with any other compounds of interest.
  • the protein-DNA complexes can also be used to detect protein-nucleic acid interactions, for example but without limitation, transcription factor-nucleic acid interactions where the target sequence of the protein-DNA complex comprises a transcription factor or fragment of a transcription factor.
  • the embodiments described above can be performed in a competitive assay format.
  • the protein vector also comprises a detectable protein sequence where 'detectable protein sequence' includes a protein sequence that is incorporated into the protein vector as a means of enabling the qualitative or quantitative measurement of the expression of the protein vector.
  • the detectable protein sequence may be a fluorescent protein such as autofluorescent protein, green fluorescent protein (GFP) or dsRed fluorescent protein.
  • the detectable protein sequence can also be used as a marker for detecting the location and/or the quantity of a protein-DNA complex.
  • the detectable protein sequence is present at, or near, the C-terminus of the protein vector such that expression of the detectable protein sequence indicates successful expression of the preceding protein sequence or sequences.
  • 'Linkers' comprise a nucleic acid sequence (or amino acid sequence) present between the nucleic acid code (or amino acid sequence) of, for example:
  • a linker can also contain one or more consensus sites permitting cleavage using a known enzyme or other molecule.
  • Cleavable linkers and their corresponding enzymes or other molecules are well known in the art, for example but without limitation, tobacco etch virus (TEV) cleavable sequences, factor X cleavable sequences and thrombin cleavable sequences.
  • a cleavable linker allows release of the target protein from its encoding DNA and assembly sequence by cleaving said linker.
  • the protein vector additionally comprises an affinity tag that is separated from the target protein by a cleavable linker
  • the affinity tagged target protein which has been released from its encoding DNA can be isolated or purified using said affinity tag; the target protein can then be further released from its affinity tag by cleavage of the linker.
  • the protein, polypeptide and/or peptide sequences comprising the protein vector are contiguous.
  • the invention provides a protein-nucleic acid complex produced according to the method of the invention.
  • the protein vector of the protein-DNA complex is attached to its encoding DNA via an assembly sequence preferably present at, or near the N-terminus of the target protein.
  • the protein vector of the protein-DNA complex is attached to its own DNA via an assembly sequence present at, or near the C-terminus of the target protein sequence.
  • the assembly sequence also comprises the target protein sequence or other sequence.
  • the target protein component of the protein-DNA complex is present (displayed) within the assembly sequence which is attached to its encoding DNA either directly or indirectly, for example via a cognate molecule.
  • streptavidin as assembly sequence within which a target protein sequence is present. Nucleic acid encoding streptavidin into which code for a target protein sequence has been inserted, is used to generate a protein vector that will subsequently assemble with its encoding DNA, said DNA being biotinylated.
  • one or more of the 8 amino acid pairs of the streptavidin core sequence forming anti-parallel strands immediately precede the side most loop of the molecule can be mutated to, for example, cysteine residues to limit any tertiary structural changes that may occur on insertion of a target protein sequence. This is possible because the distances between respective pairs of amino acids in these two anti-parallel strands ( Figure 1 indicates these two anti-parallel 8 amino acid stretches and the distance between the pairs of residues) allow cysteine substitutions without major changes to the streptavidin folding pattern.
  • the complexes of the invention can be used to identify, without limitation, target protein/protein and target protein/nucleic acid interactions as well as interactions of a target protein with other compounds of interest.
  • the detection and/or quantitation of such interactions can be achieved using conventional methods of analysing the nucleic acid component of the protein-DNA complex following their optional amplification and release from said complexes.
  • the target protein which interacts with a compound of inteerst can be identified by its association with its encoding DNA.
  • the invention provides a method for detecting the interaction of a target protein with a compound of interest comprising: (a) providing a protein-nucleic acid complex according to the method of the invention;
  • the complexes of the invention can also be presented attached to a substrate e.g. a solid or semi-solid support.
  • the complexes may therefore be provided in a spatial format as an array of protein-DNA complexes on a substantially planar surface or in wells or on beads for screening, for example, compounds of interest for binding. In this manner the complexes are are particular utility for detecting the interaction of target proteins with a compound of interest.
  • the invention provides a spatial array of protein- nucleic acid complexes produced according to the method of the invention immobilised to a support.
  • Spatial arrays can be produced using means known in the art to directly attach nucleic acids directly to a suitable substrate.
  • the protein-DNA complexes can be attached indirectly to a suitable substrate using standard means known in the art, for example but without limitation, by using nucleic acid hybridisation to immobilised complementary nucleic acids on a suitable substrate.
  • An alternative means of spatial separation can be achieved by attaching the protein-DNA complexes of the invention to beads by, for example but without limitation, hybridisation to complementary nucleic acids attached to beads.
  • the protein-DNA complexes of the invention can be used as an array to characterise antigens or other molecules or biomolecules. A target protein binding to such a compound of interest may then be identified by virtue of it being associated with the DNA encoding the protein vector comprising said target protein; the DNA may be amplified and sequenced or sequenced directly, thereby revealing the protein sequence it encodes.
  • the protein-DNA complexes of the present invention can be presented in a spatial format as an array using the affinity tag of the protein vector, wherein the affinity tag binds to a cognate sequence present on a substrate, e.g. in a spatial format as an array.
  • the assembly sequence may be an amino acid sequence recognised by an antibody which is present as an array or alternatively on a bead or other suitable substrate.
  • an array of protein-DNA complexes may be produced by binding of the affinity tag comprising, for example but without limitation, avidin, streptavidin or a derivative or fragment of such that binds to biotin where said biotin is presented in a spatial format as an array.
  • the complexes of the invention are used to produce affinity reagents.
  • the target protein sequence may be a monoclonal, a divalent or polyvalent antibody sequence, an antibody fragment or antibody mimic sequence or a complementarity determining region (CDR) or other affinity reagent sequence.
  • the affinity reagent-DNA complexes can be presented in a spatial format as an array.
  • Such an array of affinity reagent-DNA complexes can be screened for interacting molecules such as antigens or other biomolecules by, for example but without limitation, contacting an array of affinity reagent-DNA complexes with a sample of interest and detecting the interaction, again by way of example but not of limitation, by detecting a fluorescent signal such as that of a detectable protein sequence present within the protein vector.
  • the DNA component of the self-assembled protein-DNA complex is labelled for example, with a fluorescent molecule.
  • Interactions are identified for example, but without limitation, by detecting and/or quantitating the fluorescently-labelled DNA of the self-assembled protein- DNA complex.
  • the DNA of the complexes of the invention can be labelled to a high degree using well-established protocols known in the art, for example using fluorescent, radioactive or isotopic labels, or nucleic acid derivatives.
  • Suitable labelling can be achieved using for example but without limitation, primer labelling, using labelled nucleic acid analogues and derivatives during initial synthesis and/or during amplification steps if any, prior to the transcription and translation stage, labelling using PNK, DNA and RNA polymerases, DNA and RNA ligases and by chemical modification.
  • Nucleic acid labelling provides not only a means for labelling to a high specific activity, but also avoids the problem of compromising the folding, specificity and/or function of the protein component of the protein-DNA complex as protein labelling is avoided.
  • the DNA of the protein-DNA complex can be characterised by means known in the art such as electrophoresis, sequencing or hybridisation after optional amplification and release from the protein-DNA complex.
  • a protein-DNA complex where the protein component comprises an affinity reagent such as but not limited to an antibody, can be used as a cloning vehicle for said affinity reagent by performing transcription and translation to produce a quantity of protein.
  • An affinity reagent binding to such an antigen or other molecule or biomolecule can also be identified by virtue of being associated with the DNA encoding the protein vector comprising said affinity reagent; the DNA may be amplified and sequenced or sequenced directly, thereby revealing the protein sequence it encodes.
  • An alternative to presenting protein-DNA complexes is to use them in solution to screen a spatial array of biomolecules, for example but without limitation, antibody-antigen interactions, wherein an addressable array of antigens is contacted with a sample of antibody-DNA complexes.
  • the interaction can be detected as described above and the affinity reagent-DNA complex isolated and used as a cloning vehicle for the production of affinity reagent cognate for the detected target biomolecule.
  • a library of, for example but without limitation, antibody-DNA complexes in solution can be characterised and/or biomolecular targets recognised by said antibodies identified by virtue of the array being addressable.
  • the array of target biomolecules may be non- addressable.
  • the antibody-DNA complex can be amplified in situ and, by virtue of being complexed with its encoding DNA, the DNA of the affinity reagent-DNA complex can be characterised by means known in the art such as electrophoresis, sequencing or hybridisation after optional amplification and release from the protein-DNA complex, thereby revealing the protein sequence it encodes.
  • said isolated antibody-DNA complex can be used as a cloning vehicle for said antibody by performing transcription and translation to produce a quantity of antibody. It will be understood by one skilled in the art, that the above embodiments relating to affinity reagent-DNA complexes apply equally to a protein-DNA complex comprising any protein target.
  • the self-assembled protein-DNA complexes of the invention can be used as cloning vehicles for the production in a solution, i.e. not attached to a substrate, of any recombinant peptide, polypeptide or protein of interest in its native form, complexed to the DNA encoding it i.e. its own DNA.
  • the protein-DNA complexes of the invention can also be used to detect target protein/protein and target protein/nucleic acid interactions as well as interactions of a target protein with other compounds of interest if used in a spatial format as an array as described for affinity reagent-DNA complexes.
  • the protein-DNA complex of the invention is an antibody-DNA complex as described above, it is suitable for use in established immunochemical protocols, either replacing or in conjunction with ordinary polyclonal, monoclonal or recombinant antibodies (including single chain antibody Fab fragments), antibody-mimic molecules, CDRs and other protein affinity reagents.
  • immunochemical protocols include but are not limited to immunoblotting, immunoprecipitation, immunohistochemistry, immunocytochemistry, affinity chromatography, enzyme-linked immunosorbent assays (ELISA), immuno-electron microscopy etc.
  • the protein-DNA complexes of the invention provide unique advantages for immunochemical applications as each complex has a unique nucleic acid sequence associated with the protein vector.
  • nucleic acid associated with its encoded and bound protein can be performed by virtue of the fact that each DNA molecule has a unique sequence.
  • Each unique sequence can be distinguished and/or purified using nucleic acid hybridisation techniques (double helix formation by complementary nucleic acid strains or triple helix formation) or by restriction mapping, nucleic acid sequencing or other nucleic acid detection methods known in the art.
  • the complexes of the invention can be amplified using standard polymerase chain reaction (PCR) techniques well known in the art or where the complex is a protein-RNA complex, using RNA polymerase to amplify the DNA component of the complex. Coupled transcription/translation is well known in the art and kits and reagents are available commercially.
  • PCR polymerase chain reaction
  • Examples of such systems are rabbit reticulocyte lysate, wheat germ lysate, SP6/T7 in vitro T&T and RTS 100 E. Co//HY transcription and translation kits (Roche Diagnostics Ltd., Lewes, UK) and the TNT Quick coupled Transcription/Translation System (Promega UK, Southampton, UK).
  • in vitro transcription can be manipulated for the regulation of protein-DNA complex formation.
  • 3'-end modifications of DNA can pause or stall transcription, or the introduction of tertiary structure such as a triple helix can slow down transcription.
  • the processivity of an in vitro translation reaction can be manipulated for example, but without limitation, by varying the concentration of tRNAs present in a reaction mixture or by omitting a translational stop codon.
  • In vitro translation can be paused or stopped if the necessary tRNAs are not available. On addition of the missing tRNAs to a reaction mixture protein synthesis will resume.
  • This technique allows a user to manipulate the speed of protein synthesis and thus manipulate the speed of the folding of nascent protein chains. Additionally, it also permits the regulation of binding of the assembly sequence to its own DNA or suitable substrate, directly or indirectly. Alternatively, it permits the regulation of binding of the target protein sequence to other cognate sequence, for example such as amino acid motifs involved in protein-protein interactions and those motifs involved in the formation of protein complexes. For example but without limitation, translation can be paused or slowed down after the protein vector assembly sequence is produced to allow it to bind to a nucleic acid or solid support or another protein, before the complete protein is translated and released from the ribosome.
  • Translation can also be arrested by addition of a short complementary nucleic acid strand, a technique known in the art as the antisense approach (Blake, K, er a/., 1985, Biochemistry 24(22):6132-6138; Haeuptle, M., et al., Nucleic Acids Res., 1986 14(3):1427-1448; Nicole, L and Tanguay, R. Biosci. Rep., 1987, 7(3):239-246; Marcus-Sekura C. et al., Nucleic Acids Res., 1987, 15(14):5749-5763).
  • the method allows the production of self-assembled protein-DNA complexes in solution or alternatively, immobilized self-assembled protein-DNA complexes (preferably in an array format) in a single enzymatic reaction.
  • the method avoids the issue of RNA stability associated with the amount of RNA handling generally required in existing methods for the production of nucleic acid-protein complexes and hence also avoids the cross-linking problems associated with puromycin use, as described above.
  • the complexes obtained can be used for the cloning of proteins, antibodies, antibody fragments, antibody mimics and peptide aptamers.
  • Self-assembled protein-DNA complexes are especially useful in protein cloning as no additional reverse transcription step is required, unlike RNA-protein complexes.
  • the sensitivity of immunodetection at a single protein molecule level can be achieved by directly labelling the DNA of the self-assembled protein-DNA complex where the protein component of said complex comprises an affinity reagent, for example an antibody.
  • the present invention allows the production of self-assembled protein arrays using arrayed nucleic acids, a significant advantage over protein-spotting or in-situ protein synthesis arrays.
  • the protein-DNA complexes of the invention may be applied to the preparation of quantities of the encoded protein in a native, functionally correct form.
  • Panel A indicates the amino acid sequence of a fragment of Streptavidin comprising the side most loop (NTQWLLTSGTTEANAWKSTLVGHDT; SEQ ID No. 1).
  • Panel B is a representation of the secondary structure of that fragment with its secondary structure shown.
  • the side most loop links two antiparallel ⁇ -sheets. Stabilisation of the secondary structure can be achieved by substituting the amino acid pairs of the antiparallel sheet with cysteine residues.
  • the seven pairs of amino acids indicated are especially suitable due to their proximity to the loop and molecular architecture; the distances (A) are sufficient to accommodate two sulfhydryl groups and the resulting disulphide bond without major disturbances of the SAPV folding.
  • the (Trp + Gly) pair is less suitable for (Cys + Cys) substitution due to Trp involvement in biotin binding.
  • Figure 2 is the 466 base pair engineered nucleotide sequence (SEQ ID No. 2) used to make the Streptavidin-based protein vector (SAPV). Indicated are: a ribosome binding site (bold) immediately preceding the translational start codon and stop codons (underlined), the T7 terminator sequence (bold and underlined). The sequence (dotted underline) codes for the side most amino acid loop of Streptavidin selected for modification.
  • Figure 3 is the 1442 base pair engineered nucleotide sequence (SEQ ID No. 23) required to express streptavidin tagged with autofluorescent protein (AFP). Indicated are: two RNA polymerase binding sites (italic/bold), a ribosome binding site (bold) immediately preceding the translational start codon (bold/double underlined), stop codon (italic/underlined) and the linker region (underlined) separating the streptavidin and autofluorescent protein sequences.
  • Figure 4 is a histogram indicating the presence of untagged SAPV complexed with biotinylated DNA encoding tagged SAPV.
  • Panel A shows assembly of SAPV with said biotinylated DNA: lanes (a) to (d) show the 1 st to 4 th washes, respectively; lane (e) shows the eluant containing SAPV-DNA complex eluted from a protein-binding filter.
  • Panel B shows assembly of SAPV with non-biotinylated DNA encoding tagged SAPV: lanes (a) to (e) as for Panel A except that lane (e) indicates the absence of SAPV-DNA complex in the eluant.
  • Figure 5 is a histogram showing immunoprecipitation of SAPV-84.
  • the filled bar indicates normalised signal corresponding to the precipitation of the SAPV-84 on immobilised anti-BCMP84 antibody.
  • the scale is arbitrary.
  • Figure 6 is a histogram indicating the immunoprecipitation of self-assembled SAPV vectors.
  • the Streptomyces avidinii gene for streptavidin (Embl Accession No. X03591 ) was used as a scaffold for designing a SAPV.
  • Full length nucleotide sequence coding for the SAPV (Fig. 2; SEQ ID No. 2) was produced using overlapping synthetic oligonucleotides (obtained from Sigma-Genosys, Cambridge, UK) and three rounds of PCR as follows: PCR round 1 , mixture of primers used:
  • the amplified SAPV DNA fragment (Q0225) was cloned into the multiple cloning site of
  • TOPO-4-BLUNT vector The sequence of the final construct is shown in Fig. 2.
  • Expression of the SAPV protein vector was performed using a bacterial coupled transcription and translation kit obtained from Roche (RTS 100 E.coli HY). Transcription and translation reactions were performed according to manufacturer recommendations. Amounts of the
  • SAPV DNA sufficient for transcription and translation were routinely obtained by 20 cycles of
  • the SAPV was tagged with a protein tag (autofluorescent protein) for detection by Western blotting.
  • the engineered nucleotide sequence of the tagged SAPV is shown in Fig. 3 (SEQ. ID. No. 23).
  • Tagged SAPV DNA was generated as follows:
  • a linear fragment of SAPV was generated by PCR using round 3 (above) SAPV DNA as a template.
  • transcription terminator sequence 50 ⁇ l total volume, 0.5 ⁇ l 100pmol/ ⁇ l forward primer 5' tccacgctgqtcqgccacqacc ⁇ aattcqggqa ⁇ gcggaggt ⁇ 3' (SEQ ID No. 14), 5 ⁇ l 10pmol/ ⁇ l reverse primer 5' aaaaacccctcaagacccgtttagaggccccaaggggttatgctagttatcattcat tcattccaccttggtg 3' (SEQ ID No.
  • (iv) tagged SAPV was generated by joining the untagged SAPV fragment from (i) to the AFP with terminator sequence generated in (iii) as follows: 50 ⁇ l total volume, 1 ⁇ l SAPV fragment, 1 ⁇ l AFP fragment with terminator sequence, 0.5 ⁇ l each of 100pmol/ ⁇ l forward primer 5' aaattaatacgactcactat 3' (SEQ ID No. 11) and reverse primer 5' aaaaaacccctcaag accc 3' (SEQ ID No.
  • a long 5'UTR was added to the AFP-tagged SAPV as follows:
  • AFP-tagged SAPV DNA generated in part (iv) was used as a template to amplify a fragment of said DNA lacking the T7 polymerase binding site as following: 50 ⁇ l total volume, 0.5 ⁇ l 100pmol/ ⁇ l forward primer 5' agaaggagatataccat 3' (SEQ ID No. 16) and 3 ⁇ l 16pmol/ ⁇ l reverse primer 5' attaaccctcactaaaggga 3' (SEQ ID No. 17); 5 min at 96°C followed by [15 cycles of 1 min at 96°C, 30 sec at 40°C, 1.5 min at 72°C]. Final incubation was 5 min at 72°C.
  • a 5'UTR fragment was obtained from plVEX vector (Roche Diagnostics Ltd., Lewes, UK) by firstly amplifying a fragment from said vector: 50 ⁇ l total volume, 1 ⁇ l of each of 100pmol/ ⁇ l forward primer 5' aaattaatacgactcactat 3' (SEQ ID No. 11 ) and reverse primer 5' aaaaaacccctcaagaccc 3' (SEQ ID No. 12); 5 min at 96 °C followed by 15 cycles of [1 min at 96°C, 30 sec at 40 °C, 1 min at 72°C]. Final incubation was 5 min at 72°C.
  • the amplified fragment was cloned into TOPO-4-BLUNT vector and a long 5'UTR excised from said vector by PCR as follows: 50 ⁇ l total volume, 0.5 ⁇ l of 10Opmol/ ⁇ l forward primer 5' catggtatatctccttct 3' (SEQ ID No. 18) and 3 ⁇ l 16pmol/ ⁇ l reverse primer 5' caggaaacagcta tgac 3' (SEQ ID No. 19); 5 min at 96°C followed by 15 cycles of [1 min at 96°C, 30 sec at 40°C, 30 sec at 72°C]. Final incubation was 5 min at 72°C.
  • template DNA a mixture of said tagged SAPV DNA and DNA containing the 5'UTR fragment generated in part (vi), 50 ⁇ l total volume, 0.5 ⁇ l of each of 100pmol/ ⁇ l forward primer 5' aaattaatacgactcactat 3' (SEQ ID No. 1 1 ) and reverse primer 5' aaaaaacccctcaagaccc 3' (SEQ ID No.
  • the PCR product containing tagged SAPV DNA from part (vii) was purified by ethanol precipitation, prior to use in the transcription and translation reaction. A range of DNA concentrations and transcription and translation reaction temperatures were used in the generation of tagged Streptavidin. Reactions were typically run overnight. Detection of the tagged SAPV on Western blots was performed using anti-GFP Rabbit polyclonal antibody (AbCam Ltd, Cambridge, UK). Aliquots (5 ⁇ l of 20 ⁇ l) of each of the coupled transcription and translation reactions were loaded onto a precast 4-12% NuPAGE gel (Invitrogen).
  • the proteins were resolved by SDS-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membrane (0.2 ⁇ M pore size, Invitrogen) using an Xcell SureLock Minicell and Blot Module according to the manufacturer's instructions.
  • the membrane was blocked for 1 hr. in Tris buffered saline plus 0.1%(v/v) Tween-20 (TBST) with 2%(w/v) powdered milk and probed with a 1:3000 dilution of AbCam rabbit anti-GFP polyclonal (1hr. room temperature; RT) in TBST/milk.
  • the membrane was washed in TBST (3 times for 5-10 min.
  • the optimal experimental conditions for coupled transcription and translation reactions selected from the results of the above experiment use 2 ⁇ g DNA per 20 ⁇ l reaction volume with the synthesis temperature maintained at 21 °C.
  • the following experiment shows one example of assembling a complex of untagged SAPV with its encoding DNA and is in no way intended to be limiting.
  • Untagged SAPV DNA lacking stop codons was obtained by PCR using tagged SAPV DNA generated as in part (vii) as a template as follows: 50 ⁇ l volume, 3 ⁇ l of 16pmol/ ⁇ l forward primer 5' gtaaacgacggccag 3' (SEQ ID No. 13) and 0.5 ⁇ l of 100pmol/ ⁇ l reverse primer 5' ttccaccttggtgaaggtgtcgtggccgaccagcgtggacttccaggcgttggcctcggtggtgcggaggtca gca 3' (SEQ ID No. 9); 6 min at 96°C followed by 15 cycles of [1 min at 96°C, 30 sec at 40°C, 1 min at 72 °C]. Final incubation was 5 min at 72°C.
  • Biotinylated and non-biotinylated SAPV DNA was generated by PCR using either 5'-biotinylated or non-biotinylated primers from Sigma- Genosys (Cambridge, UK): forward primer: 5' aaattaatacgactcactat 3' (SEQ ID No. 11); reverse primer: 5' aaaaacccctcaagaccc 3' (SEQ ID No. 12).
  • Eluted DNAs were detected by PCR as follows: 10 ⁇ l of each of the wash through and eluates from each assembly reaction was amplified in parallel using forward primer 5' aaattaatacgactcactat 3' (SEQ ID No. 11); reverse primer: 5' aaaaaacccctcaagaccc 3' (SEQ ID No. 12); 35 cycles of [1 min at 96°C, 30 sec at 40°C, 1.5 min at 72°C] were performed. Amplified products were separated on 2.5%(w/v) agarose gels containing EtBr. Equal amounts of PCR products were loaded onto each lane (see Figure 4).
  • a display system using SAPV is described.
  • a loop in the amino acid sequence of Streptavidin has been selected ( Figure 1 ) as a useful site for insertion of a target protein sequence or for the insertion of other protein fragments, peptides or tags.
  • the core protein sequence of the Streptavidin and the Streptavidin-based SAPV contains a 9 amino acid long loop ( Figure 1 ) which was found to be mostly suitable for modifications, such as SAPV extension, modification or for expressing of other proteins, peptides and tags. This choice is based on the Streptavidin molecular architecture ( Figure 1).
  • SAPV nucleic acid sequence was modified to display peptide fragments of albumin and BCMP84 (see PCT Application No. WO 01/62914) proteins as follows:
  • STOP codons were added to both constructs as follows: SAPV-84 or SAPV-alb5 DNA fragments (as described above) were used as a template in PCR amplifications. Conditions: 50 ⁇ l total volume, 1 ⁇ l of 10Opmol/ ⁇ l forward primer 5' gtaaaacgacggccag 3' (SEQ ID No.
  • a quantity of DNAs encoding each of SAPV-84 and SAPV-alb5 were produced for transcription and translation by PCR: forward primer 5' aaattaatacgactcactat 3' (SEQ ID No. 11); reverse primer: 5' aaaaaacccctcaagaccc 3' (SEQ ID No. 12).
  • a coupled transcription and translation reaction (50 ⁇ l volume) was run as described above (see Examples 2 and 3 for details) using DNA coding for untagged SAPV-84. Assembly was achieved by overnight incubation at 4°C of biotinylated SAPV-84 DNA construct with the coupled transcription and translation reaction supernatant as before.
  • the SAPV-DNA complex was immunoprecipitated with either anti-BCMP84 or anti-albumin antibodies as follows: 3-mm squares of wetted nitrocellulose membrane were incubated with either 20 ⁇ l of anti-albumin antibody or 20 ⁇ l of anti-BCMP84 antibody.
  • the squares were washed twice in 1 ml PBS then blocked by incubation in 2% (w/v) powdered milk plus 0.1 mg/ml tRNA in PBS for 30 min. Blocking buffer was removed and the nitrocellulose membrane squares were incubated for 60min. with the assembled SAPV-DNA complex. The squares were washed three times in wash buffer (1 ml PBS supplemented with 0.01 mg/ml tRNA and 2%(w/v) powdered milk).
  • the nitrocellulose membrane squares were incubated with 50 ⁇ l elution buffer (80mM glycine, pH 2.45) supplemented with 0.4%(w/v) powdered milk and 0.01 ⁇ g/ml tRNA.
  • the eluted samples were neutralised by the addition of 11 ⁇ l 2M NaOH.
  • the presence of the SAPV-84 in the eluted samples was detected by PCR amplification of the SAPV-84 DNA expression vector as follows: forward primer 5' aaattaatacgactcactat 3' (SEQ ID No. 11) and reverse primer 5' aaaaacccctcaagaccc 3' (SEQ ID No. 12).
  • the following example demonstrates self-assembly of SAPV-84 with its encoding DNA and of SAPV with an albumin peptide as a target sequence (SAPV-alb5) inserted into the side most loop with its encoding DNA.
  • An "empty" SAPV was included as a control. Coupled transcription and translation (using RTS 100 E.coli HY kit from Roche ) and self- assembly of the SAPV-84, SAPV-Alb5 and an "empty" SAPV-only protein vectors were performed as before.
  • each reaction contained approximately 16 ⁇ g of biotinylated SAPV DNA expression vectors (either SAPV-84, SAPV-alb5 or an "empty" SAPV-only DNA expression vector) in a total volume of 100 ⁇ l each.
  • Two 40 ⁇ l aliquots of cleared supernatant from each self-assembly reaction were transferred to fresh tubes.
  • the SAPV-84 and SAPV-alb5 were precipitated from these aliquots by incubation with either anti-BCMP84 or anti-albumin antibodies as follows:

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Abstract

La présente invention concerne des techniques de production de protéines de recombinaison qui s'auto-assemblent en complexes avec des acides nucléiques en solution ou sur des supports et des complexes acide nucléique-protéine produits selon cette technique, par exemple dans une structure de jeu ordonné d'échantillon.
PCT/GB2002/003351 2001-07-24 2002-07-23 Complexes acide nucleique-proteine WO2003010176A2 (fr)

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