WO2006082058A2 - Cell-free translation system for the production of (chemically) modified proteins - Google Patents

Cell-free translation system for the production of (chemically) modified proteins Download PDF

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WO2006082058A2
WO2006082058A2 PCT/EP2006/000926 EP2006000926W WO2006082058A2 WO 2006082058 A2 WO2006082058 A2 WO 2006082058A2 EP 2006000926 W EP2006000926 W EP 2006000926W WO 2006082058 A2 WO2006082058 A2 WO 2006082058A2
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cell
release factor
translation system
free translation
residue
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PCT/EP2006/000926
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French (fr)
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WO2006082058A3 (en
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Mathias Sprinzl
Dmitry Agafonov
Kersten Rabe
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Universität Bayreuth
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    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/16—Purine radicals
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria

Definitions

  • the present invention relates to a cell-free translation system comprising a nonsense-codon suppressing agent and an anti-release factor antibody which precipitates and/or crosslinks a release factor in said cell-free translation system.
  • the present invention further relates to a kit comprising said cell free translation system.
  • the present invention relates to an anti-release factor antibody directed against release factor 1 of Thermus thermophiles and, inter alia, obtainable by eliciting an in vivo humoral response against release factor 1 from Thermus thermophilus or a fragment thereof in a non-human vertebrates.
  • the present invention relates to the use of an anti-release factor antibody for the preparation of a cell-free translation system.
  • the present invention relates to a method for the production of an anti-release factor antibody comprising the steps of eliciting an in vivo humoral response against release factor 1 from Thermus thermophilus or a fragment thereof in a non-human vertebrate. Furthermore, the present invention provides for a method for the production of alloproteins, comprising the step of translating RNA into a translation product in the cell-free translation system of the present invention.
  • the present invention relates to a puromycin derivative having a residue covalently attached directly or via a linker to one of its elements selected from the group consisting of the element N 4 of the cytosine- residue of an 5' attached cytidine-residue and the element C 5 of the cytosine-residue of an 5' attached cytidine-residue.
  • the present invention relates to the use of a puromycin derivative having a residue covalently attached directly or via a linker to one of its elements selected from the group consisting of the element N 4 of the cytosine-residue of an 5' attached cytidine-residue and the element C 5 of the cytosine-residue of an 5' attached cytidine-residue, in the cell-free translation system of the present invention, in the method for the production of alloproteins of the present invention.
  • the invention also provides for specific kits described herein. Protein conjugates, e.g. glycoproteins, nucleoproteins, phosphoproteins and lipoproteins, are involved in numerous cellular functions.
  • nucleic acid with a specific sequence to proteins allows to link properties of these two distinct groups of biopolymers in one molecule.
  • the resulting conjugates can be used for variety of applications including preparation of synthetic enzymes (Corey (1987), Science, 238, 1401-1403), gene therapy (Zanta (1999), Proc. Natl. Acad. Sci. U.S.A., 96, 91-96), construction of protein microarray (Niemeyer (1994), Nucleic Acid Res., 22, 5530-5539), creation of molecular scale devices (Keren (2002), Science, 297, 72-75), and development of immunological assays (Niemeyer (2003), Nucleic Acids Res., 31 , e90).
  • a specific conjugation of proteins with variety of functional groups can be achieved by ligation of chemically synthesized modified peptides with target polypeptides leading to recombinant proteins (Cotton (1999), Chem. Biol., 6, R247-R256) (Goody (2002), Chembiochem., 3, 399-403) (Eisele (1999), Bioorg. Med. Chem., 7, 193-224).
  • recombinant proteins containing C-terminal ⁇ -thio-ester can be conjugated with cysteinyl- deoxyribonucleic acids by protein ligation leading to oligonucleotides specifically attached to C-terminus of recombinant protein (Takeda (2004), Bioorg. Med. Chem. Lett., 14, 2407-2410).
  • protein conjugates e.g., protein-nucleic acid conjugates
  • a convenient and universal covalent conjugation procedure was not yet developed. This problem was addressed for several times within the art.
  • One approach aimed the suppression of nonsense-codons by a suppressor tRNA, which is aminoacylated by an unnatural amino acid during in vitro translation, and thereby covalently attaching different amino acids to the synthesized proteins.
  • the suppression of the nonsense-codons was either achieved by the expansion of the genetic code (Wang (2002), Chem. Commun. (Camb). (1):1-11) or by the use of the low molecular weight antibiotic puromycin.
  • one of the three available triplets coding for termination of translation are "recorded” for recognition by suppressor tRNAs that are able to read one of these stop codons.
  • the UAG codon that is recognized by the release factor 1 (RF 1) is selected for recognition by a suppressor tRNA (tRNA Su P) chemically aminoacylated, i.e. not enzymatically, by an unnatural amino acid.
  • tRNA Su P suppressor tRNA
  • puromycin was incorporated into the C-terminus of the protein and is used as a vehicle to transfer a variety of functional groups into the (in vitro) synthesized protein.
  • Biosynthesis of proteins in all living organisms is based on peptidyl transferase reaction catalyzed by the ribosome.
  • a nucleophilic attack on the carbonyl group of the ester bond of the peptidyl-tRNA by the amino group of the aminoacyl-tRNA results in formation of an amide (peptide) bond (Fig. 1A).
  • the antibiotic puromycin that by its structure resembles the aminoacylated 3'-terminal adenosine of the aminoacyl-tRNA (Fig. 1 B), can compete with aminoacyl-tRNA.EF-Tu.GTP ternary complex for the ribosomal A-site.
  • puromycin-mediated termination is, however, very low.
  • the technical problem underlying the present invention is the provision of means and methods for the amelioration of translation efficiencies in particular the translation efficiencies for the production of (chemically) modified proteins and/or alloproteins.
  • the present invention relates to a cell-free translation system comprising a nonsense-codon suppressing agent and an anti-release factor antibody which precipitates and/or crosslinks a release factor in said cell-free translation system.
  • the present invention solves the above identified technical problem since, as documented herein below and in the appended examples, the cell-free translation system of the present invention provides for at least 50% suppression, more preferably at least 60% suppression, more preferably at least 70% suppression, more preferably at least 80% suppression, even more preferably at least 90% suppression and most preferably at least 98% suppression. As shown in the appended examples, the present invention even provides for a cell-free translation system, wherein the nonsense-codons are completely suppressed, i.e. up to nearly 100% suppression can be achieved.
  • Suppression of nonsense-codons can be measured as shown in the appended examples, in particular by detecting the incorporation of nonsense-codon suppressing agents in proteinaceous material obtained in said in-vitro translation systems.
  • nonsense-codon suppressing agents in proteinaceous material obtained in said in-vitro translation systems.
  • An example may be the incorporation of radioactively labelled puromycin or derivatives thereof.
  • the inventive use of anti-release factor (RF) precipitating and/or crosslinking antibodies provides for nearly 100% suppression of nonsense-codons.
  • the use of aptamers directed against the same release-factor only provides for a portion of such a suppression, even if aptamers are present in excess.
  • the reported and measured efficiency of translation systems of the prior art lays in values of about up to 30 % suppression of nonsense-codons.
  • anti-RF antibodies i. e. antibodies capable of precipitation and/or crosslinking a RF leads to highly-efficient suppression of nonsense-codons (by nonsense-codon suppressing agents) and, thereby, to efficient in vitro synthesis values.
  • the present invention provides, inter alia, for a system for coupled in vitro protein synthesis, capable for specific and highly-efficient production of proteins, in particular of proteins comprising chemical modifications and/or additions (C-terminal and/or internal).
  • the system provided herein is particularly useful in the synthesis of (chemically) modified proteins and/or alloproteins.
  • protein(s) also includes “peptide(s)” or “polypeptide(s)”.
  • the meaning of the terms “protein(s)”, “peptide(s)” or “polypeptide(s)” are well known in the art (see, e.g., Stryer (1995), Biochemistry, 4 th edition).
  • the term “peptide” comprises joined amino acid residues, whereby the alpha-carboxyl group of one amino acid is joined to the alpha- group of another amino acid by a peptide bond (amide bond); see also Stryer ((1995), loc. cit.).
  • peptide(s) comprises any such joined amino acid residues, whereby at least three, preferably at least five, most preferably at least seven amino acids (amino acid residues) are linked via said peptide bond (amide bond).
  • polypeptide comprises, in accordance with this invention, at least 15 joined amino acid residues, more preferably at least 20 amino acid residues. Accordingly, joined amino acid residues comprising 3 to 14 amino acid residues are to be considered in accordance with this invention as "peptide” whereas joined amino acid residues comprising 15 or more amino acid residues are considered as polypeptides.
  • protein is used as synonym with the term “polypeptide”, whereas the term “protein” also may comprise a specific biological, biochemical or pharmaceutical function exerted by said protein.
  • a protein is a polypeptide.
  • the terms “protein”, “peptide” and “polypeptide” also comprise molecules comprising at least one unnaturally occurring amino acid residue or at least one unusual amino acid residue and is not limited to proteinaceous structures comprising the twenty normally occurring amino acid residues; see also Stryer ((1995), loc. cit.).
  • nonsense-codon suppressing agents for example the antibiotic puromycin and/or suppressor tRNA.
  • nonsense-codon suppressing agents can be used as vehicles to carry chemical residues (for example (unnatural) amino acids, reporter groups, affinity tags or even large oligomeric structures) to be linked with a polypeptide.
  • nonsense-codon suppressing agents can also be employed to efficiently carrying chemical residues to proteinaceous structures (proteins) synthesized. Said "chemical residues” can then be efficiently covalently linked to the protein structure. Accordingly, with the present invention, "unnatural" proteins and/or alloproteins may be successfully synthesized.
  • the present invention allows to use conventional coupled transcription/translation systems as a basis for high yield production of alloproteins.
  • the conventional systems have, in accordance with this invention, to be further modified by a) the addition of an anti-RF antibody (for example a serum directed against the corresponding RF) and b) the addition of nonsense-codon suppressing agents.
  • an anti-RF antibody for example a serum directed against the corresponding RF
  • nonsense-codon suppressing agents for example a serum directed against the corresponding RF
  • nonsense-codon suppressing agent as used herein is known in the art and relates to an agent that is capable to bind to the A-site of a ribosome programmed by a stop codon. Said stop codons are known in the art and may be UAA, UAG or UGA, preferably, UAG.
  • the nonsense-codon suppressing agent itself may be covalently bound to the elongating peptide-chain or may be delivering a substance that is bound to the elongating peptide chain. Said nonsense-codon suppressing agent may prevent normal termination accomplished by release factors or termination factors or said nonsense-codon suppressing agents may replace normal termination accomplished by release factors or termination factors.
  • said nonsense-codon suppressing agent that delivers a substance to be bound to the elongating peptide-chain prevents normal termination.
  • the nonsense-codon suppressing agent, delivering the substance to be bound to the polypeptide-chain may be a aminoacyl- tRNA, preferably a suppressor aminoacyl-tRNA, more preferably a suppressor aminoacyl-tRNA (CUA) .
  • the nonsense-codon suppressing agent to be covalently bound to the polypeptide chain may be, inter alia and preferably, puromycine or a derivative thereof as defined herein below.
  • anti-RF antibody in particular "anti-RF1 antibody” as employed herein refers to an antibody, a plurality of antibodies and/or a serum comprising such antibodies which is/are able to specifically bind to, interact with and/or detect RFs, preferably RF1 , more preferably RF1 from E. coli (e.g. as shown in SEQ ID NO: 6) or a fragment thereof.
  • said "anti-RF antibody” must be capable of precipitating (in the in vitro system) the RF and/or must be capable of crosslinking said RF.
  • the "precipitation” and/or crosslinking” leads to an inactivation of the RF, inter alia, due to the formation of larger RF- antibody complexes.
  • precipitates and/or crosslinks refers to the capability of an anti-release factor antibody to bind and to inactivate a release factor. Therefore, said binding leads to an inactivation of said release factors which is equivalent of a depletion of said release factor (from cell-free translation systems).
  • activation refers to making said release factors incapable to bind to the A-site of the ribosome and thereby incapable to cause termination of the peptide-chain and its release from the ribosomal complex.
  • the precipitating and/or deactivating activity of anti-RF antibodies can, inter alia, be measured by the residual RF activity in the in vitro translation system after adding of said anti-RF antibodies, by testing the hydrolysis of a peptide from peptidyl-tRNA located in the P-site (Freistroffer (2000), Proc Natl Acad Sci U S A. 97, 2046-51) or by a gel electrophoresis followed by Western blotting, which is a common laboratory praxis.
  • the formation of complexes between the RFs and anti-RF antibodies can be measured by measuring complex formation, for example in native gels.
  • individual proteins/polypeptides/peptides and complexes may be detected in form of either single individual bands or in form of complexes-formed broader bands in high molecular weight regions.
  • Said banding pattern may be compared in gels comprising reducing agents (like DTT) and in gels without such agents.
  • the corresponding technology is standard and known in the art; see, e.g. Sambroock (2001. Molecular Cloning, Ed 3.).
  • the precipitating and/or deactivating activity of anti-RF antibodies can be measured by complementing the depletion of RF through said antibodies or sera by addition of certain amounts of RF protein. Such a measurement method is exemplified in the appended examples (Example 20, Figure 21).
  • Corresponding antibodies may easily be prepared as demonstrated in the appended examples and as known in the art.
  • Said antibodies and/or sera may, inter alia, be prepared by immunization of a non-human vertebrate with purified and/or recombinants produced "release factors".
  • release factors for example a polyclonal serum against release factor 1 (RF1) of Thermus thermophilus (T. th; SEQ ID NO: 2) can be routinely and reliably prepared.
  • RF1 polyclonal serum against release factor 1
  • T. th Thermus thermophilus
  • anti-RF antibody also relates to a serum, in particular a purified serum, i.e. a purified polyclonal serum.
  • the antibody molecule is preferably a full immunoglobulin, like an IgG, IgA, IgM, IgD, IgE, IgY (for example in yolk derived antibodies).
  • antibody as used in this context of this invention also relates to a mixture of individual immunoglobulins.
  • the antibody/antibody molecule is a fragment of an antibody, like an F(ab), F(abc), Fv Fab' or F(ab) 2 .
  • antibody as employed in the invention also relates to derivatives of the antibodies which display the same specificity as the described antibodies. Such derivatives may, inter alia, comprise chimeric antibodies or single-chain constructs. Yet, most preferably, and as shown in the examples, said "anti-RF antibody” relates to a serum. Also a purified (polyclonal) serum and, preferably, to a non-purified crude polyclonal serum.
  • the antibody/serum is obtainable, and preferably obtained, by the method described herein and illustrated in the appended examples or by other methods known in the art.
  • said anti-RF antibody in particular said anti- RF1 antibody, may specifically deplete one particular RF (e.g. RF1 (e.g. having the amino acid sequence of SEQ ID NO: 6)) keeping (an-)other RF(s) (e.g. RF2 (e.g. having the amino acid sequence of SEQ ID NO: 8)) active.
  • RF1 e.g. having the amino acid sequence of SEQ ID NO: 6
  • an-other RF(s) e.g. RF2 (e.g. having the amino acid sequence of SEQ ID NO: 8)
  • a nonsense- codon suppressing agent e.g. suppressor tRNA
  • STOP-codon e.g. UAG
  • the first RF e.g. RF1
  • the second RF e.g.
  • Said first STOP-codon may be an artificial STOP-codon lying inside of the open reading frame of a mRNA to be translated.
  • Said second STOP- codon may lie at the end of said open reading frame.
  • release factor as used herein relates to any factor(s) that is/are capable to bind to the A-site of a ribosome programmed by a stop codon, whereby the stop codon is defined as mentioned herein above.
  • said release factor causes termination of the elongation of a peptide-chain during translation process, and thereby leads to a release of the nascent peptide-chain from the ribosomal complex.
  • release factor refers to release factors that are contained in cell-free translation systems.
  • release factor also relates to a fragment of a release factor as defined herein.
  • fragment (of a release factor) as used herein relates to fragments of a length of at least 30, at least 40, at least 50, more preferably at least 60, ever more preferably at least 65 amino acid residues of a (native) RF as defined herein.
  • the amino acid sequence of RFs are known in the art and also specified herein below.
  • said fragment comprises at least such stretch of amino acids that (polyclonal) antibodies may be raised against this fragments and that these obtained antibodies are capable to precipitate and/or crosslink a release factor in a cell-free translation system.
  • cell-free translation system refers to cell-free translation systems commonly employed in in vitro synthesis approaches for proteins.
  • cell-free translation system are known in the art (e.g. Spirin (1990), American Society for Microbiology, 56-70; Stiege (1995), J. Biotechnol. 41:81-90; Zubay (1973), Imm. Rev. Genet. Vol. 7, page 267; Pelham (1976), Eur. J. Biochem. Vol. 131 , page 289; WO 9307287; EP 1254962; US 5571690; EP 1251168) and also further defined herein below.
  • cell-free translation systems known do not comprise an anti-release factor antibody and do not comprise a nonsense-codon suppressing agent. Accordingly, known cell-free translation systems do not comprise the two essential parts of the cell-free translation system of the present invention a) an anti-RF antibody capable of inactivating the release factor and b) a nonsense-codon suppressing agent.
  • one advantage of the invention is that an effective attachment of an (additional) substituent to the nascent peptide chain can be obtained.
  • Cell-free translation systems are made from cell-free extracts produced from prokaryotic or eukaryotic cells that contain all the necessary components to translate RNA (in particular mRNA) into protein.
  • Cell-free extracts can be prepared from prokaryotic cells such as E. coli cells (e.g. Zubay (1973), Imm. Rev. Genet. Vol. 7, page 267) and from eukaryotic cells such as rabbit reticulocytes (e.g. Pelham (1976), Eur. J. Biochem. Vol. 131, page 289) and wheat germ cells (e.g. Spirin (1990), American Society for Microbiology, 56-70; Stiege (1995), J. Biotechnol. 41:81-90).
  • prokaryotic cells such as E. coli cells (e.g. Zubay (1973), Imm. Rev. Genet. Vol. 7, page 267) and from eukaryotic cells such as rabbit reticulocytes (e.g. Pelham (1976), Eur. J. Biochem.
  • RF1 specific, inactivating antibodies to cell free translation systems preferably to cell free translation systems of prokaryotic origin (e.g. coupled in vitro transcription/translation systems derived from E. coli) leads to dramatic increase of puromycin derivative (e.g. [ 32 P]pGpCpPuromycin or other 5'- modified puromycin derivatives) incorporation into the C-terminus of a protein (e.g. of a full-length esterase), while leaving elongation unaffected.
  • puromycin derivative e.g. [ 32 P]pGpCpPuromycin or other 5'- modified puromycin derivatives
  • This approach allows the use of (a) conventional coupled transcription/translation system(s) as base for high- yield production of modified proteins, in particular protein-puromycin conjugates.
  • the methodology of the present invention allows the covalent conjugation of (a) chemical structure(s) including different dyes, affinity tags, spin labels etc. on the C-terminus of a protein to be synthesized with high yield by the cell-free translation system provided herein and the methods disclosed in this invention.
  • puromycin modified with an azide group could be used for subsequent one site addressed Staudinger reaction (K ⁇ hn (2004), Angew. Chem. Int. Ed Engl., 43, 3106-3116) and utilization of puromycin carrying ⁇ -thio-ester group may allows to use the protein ligation technology (Lovrinovic (2003), Chem. Commun. (Camb. ), 822-823).
  • This conjugation technology can be used to develop concepts for preparation of protein arrays and novel tools to study protein interactions (Ramachandran (2004), Science, 305, 86-90).
  • Puromycin derivatives modified with an azide group may also by used for site-specific protein immobilisation, e.g. by taking advantage of the Staudinger ligation (Soellner, 2003, J.Am.Chem.Soc. 125, 11790-11791.).
  • the experiments effected herein demonstrate that in particular puromycin can be used as a delivery carrier of different chemical functional groups for specific C- terminal labelling of protein in high yield. It is further demonstrated that puromycin and corresponding derivatives can be safely used as a nonsense-codon suppressing agent comprised in the cell-free translation system of the present invention.
  • a particular protein namely the Esterase 2 from Alicyclobacillus acidocaldarius (Est2) can be safely and efficiently produced from a template comprising an (artificially) introduced internal stop codon.
  • the coding sequence of said esterase 2 to be employed in terms of the present invention is shown in SEQ ID NO: 38, the corresponding amino acid sequence is shown in SEQ ID NO: 39 or SEQ ID NO. 55.
  • the amino acid sequence of said esterase 2 is that of SEQ ID NO. 55.
  • SEQ ID NO: 39 the internal methionine residues (Met (M)), encoded by their corresponding nucleotide residues of SEQ ID NO: 38, are indicated as X.
  • Met (M) methionine residues
  • est2 mRNA(amber 155) as a template and the possibility to deactivate the endogenous RF1 in the in vitro translation system by RF1 antibodies permits an optimal adjustment of RF1 and tRNA Ser(CUA) concentrations to achieve a complete suppression and at the same time a maximal retention of enzymatic activity of the esterase.
  • a preferred cell-free translation system of the present invention or to be employed in context of this invention is a cell-free coupled transcription/translation system.
  • “Coupled” in context of the present invention means that the transcription and translation occur concurrently in one reaction.
  • “Coupled” in the sense of the present invention can also mean that the mRNA molecules which have just been formed by transcription are already translated by the ribosomes.
  • “Coupled” in context of the present invention can also mean that the transcription and translation occur simultaneously after the addition of DNA to the extract.
  • the use of RNA as a template in E. coli extracts results in protein production but such a reaction is not called “coupled”.
  • said cell-free translation system may be of prokaryotic and/or eukaryotic origin, yet preferring it is of prokaryotic origin.
  • a cell-free translation system of E. coli origin may be employed.
  • Prokaryotic cell-free translation systems in particular from E. coli, can be used when the gene to be expressed has been cloned into a vector containing the appropriate prokaryotic regulatory sequences, such as a promoter and ribosome binding site.
  • the cell-free coupled transcription/translation systems may (further) comprise the following ingredients:
  • ribonucleotide triphosphates like ATP, CTP, GTP, UTP, etc.
  • - amino acids or a mixture of amino acids to be incorporated in a nascent peptide, polypeptide or peptide, in particular leucine.
  • Said systems may comprise also aminoacyl-tRNAs as defined herein or as generally known in the art.
  • Said aminoacyl-tRNAs may originally be comprised in the cell-free extract. But also additional tRNAs, like suppressor seryl-tRNA SER(CUA) may be added.
  • the magnesium ions that are contained in the cell-free coupled transcription/translation systems of the invention are at a concentration at which RNA is transcribed from DNA and RNA translates into protein. More preferably, the magnesium ions are in form of MgCI 2 , e.g. at a concentration of 9-12 mM.
  • the cell-free coupled transcription/translation systems may comprise the ingredients as listed below:
  • each amino acid can be applied as a 14C amino acid with a concentration of 100 ⁇ M (e.g. 14C-leucine))
  • aprotinin 10 ⁇ g/ml reaction mixture leupeptin 5 ⁇ g/ml reaction mixture, pepstatin 5 ⁇ g/ml reaction mixture; and
  • cell-free coupled transcription/translation system is merely an illustrative example of a cell-free system to be employed in context of this invention.
  • An inventive cell-free translation system also comprises a) the inactivating anti-RF antibodies and/or sera and b) the nonsense-codon suppressing agent as defined herein. Corresponding examples are also given in the experimental part.
  • cell-free translation systems in particular cell-free coupled transcription/translation systems is well known in the art. Said systems are also commercially available, e. g. from Promega GmbH or Roche Diagnostics GmbH (Mannheim, Germany). Most preferably, and also shown in the experimental part, said cell-free coupled transcription/translation systems may be comprised in evaluation size transcription/translation kits purchased from RiNA GmbH (Berlin, Germany).
  • the cell-free translation systems to be employed in context of the present invention may (further) comprise a labelled amino acid.
  • a labelled amino acid By incorporation of said labelled amino acid, it is possible to monitor and/or track the synthesis of a protein or to identify the location (e.g. in a polyacrylamide gel) of said protein.
  • the labelled amino acid is a radioactively labelled amino acid, more preferably the labelled amino acid is [ 14 C]leucine, [ 14 C]valine and/or [ 14 C]isoleucine, most preferably the labelled amino acid is [ 14 C]leucine.
  • the cell-free translation system of the present invention may also be of eukaryotic origin.
  • a wheat germ extract cell-free translation system or a rabbit reticulocyte lysate cell-free translation system would be preferred, but a cell-free translation system based on lysates from oocytes or eggs (e.g. oocytes from Xenopus) may be also applicable.
  • eukaryotic systems may preferably be used for the expression of eukaryotic genes or mRNA and are also well known in the art.
  • Another preferred cell-free translation system to be employed in the context of this invention is a cell-free translation system, wherein said nonsense-codon suppressing agent is puromycin or a derivative thereof and/or a suppressor aminoacyl-tRNA.
  • Said suppressor tRNA may be, e.g. suppressor seryl-tRNA Ser (CUA) .
  • the nonsense-codon suppressing agent may also be e.g. selected from the group consisting of:
  • the residue to be covalently attached to the puromycin may be selected from the group consisting of nucleic acids like DNA, RNA, locked DNA, PNA, oligonucleotide-thiophosphates and substituted ribooligonucleotides and other nucleic acids. It is also envisaged that other residues, like peptides or "tags" can be attached to said puromycin to be integrated in a protein during its in vitro synthesis. Further and/or additional modifications on the puromycin structure, in particular of the puromycin-derivatives to be employed and as disclosed herein, are also envisaged.
  • the puromycin (-derivatives) to be employed in context of the present invention and as disclosed herein may be labelled, preferably at the 5'-End.
  • Said labelling preferably is with a radioactive element, more preferably with 32phosphorus (32P), most preferably with a phosphate group containing 32phosphorus ([32P]p).
  • nucleic acid(s) and/or nucleic acid molecule(s) encompasses all forms of naturally occurring types of nucleic acid(s) and/or nucleic acid molecules as well chemically synthesized nucleic acids and also encompasses nucleic acid analogs and nucleic acid derivatives such as e. g. locked DNA, PNA, oligonucleotide tiophosphates and substituted ribo-oligonucleotides.
  • nucleic acid and/or “nucleic acid molecules(s) also refers to any molecule that comprises nucleotides or nucleotide analogs.
  • nucleic acid(s) and/or “nucleic acid molecule(s)” refers to oligonucleotides or polynucleotides, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).
  • the "nucleic acids” and/or “nucleic acid molecule(s)” may be made by synthetic chemical methodology known to one of ordinary skill in the art, or by the use of recombinant technology, or may be isolated from natural sources, or by a combination thereof.
  • the DNA and RNA may optionally comprise unnatural nucleotides and may be single or double stranded.
  • Nucleic acid(s) and/or “nucleic acid molecule(s)” also refers to sense and anti-sense DNA and RNA, that is, a nucleotide sequence which is complementary to a specific sequence of nucleotides in DNA and/or RNA.
  • nucleic acid(s) and/or “nucleic acid molecule(s)” may refer to DNA or RNA or hybrids thereof or any modification thereof that is known in the state of the art (see, e.g., US 5525711 , US 4711955, US 5792608 or EP 302175 for examples of modifications).
  • Such nucleic acid molecule(s) are single- or double- stranded, linear or circular, natural or synthetic, and without any size limitation.
  • the nucleic acid molecule(s) may be genomic DNA, cDNA, mRNA, antisense RNA, ribozyme or a DNA encoding such RNAs or chimeroplasts (Colestrauss (1996)).
  • nucleic acid molecule(s) is/are in the form of a plasmid or of viral DNA or RNA.
  • Nucleic acid molecuie(s) may also be oligonucleotide(s), wherein any of the state of the art modifications such as phosphothioates or peptide nucleic acids (PNA) are included.
  • the residue, covalently attached to said puromycin or said derivative thereof may be selected from the group consisting of a Cy3-fluorosphore, biotin or an other affinity tag, a reactive group for affinity labelling or any other reporter group (for review see Gilmore (1999), Topics in Current Chemistry, 202, 77- 99).
  • the reactive group for instance, can be an azide group to use for subsequent one site addressed Staudinger reaction (K ⁇ hn (2004), Angew. Chem. Int. Ed Engl., 43, 3106-3116) or an ⁇ -thio-ester group to use for the protein ligation technology (Lovrinovic (2003), Chem. Commun. (Camb.), 822-823).
  • puromycin derivatives modified with an azide group may also by used for site-specific protein immobilisation, e.g. by taking advantage of the Staudinger ligation (Soellner, 2003, loc. cit.).
  • the residue, covalently attached to said puromycin or said derivative thereof may be also be a(n) (other) spectroscopic reporter.
  • Jinker refers to a molecule capable to connect said puromycin (-derivative) and said residue covalently.
  • the linker between the puromycin (-derivative) and said residue may be an aliphatic amine derivative, preferably forming an amide with a fatty acid attached to a polyoxyamine.
  • said linker may comprise the following molecule:
  • part of the molecule indicated in squared brackets may be of different length, e.g. may be elongated by additional or shortened by less carbon residues and/or oxygen residues.
  • (n) is at least 3, preferably at least 5 carbon residues. Yet, the amount of carbon residues (n) may most preferably be 5 or 9.
  • linker between the puromycin (-derivative) and said residue may act as a
  • Nonsense-codon suppressing agent are known in the art, as documented above. However, the "nonsense-codon suppressing agent” comprised in a cell-free translation system of the present invention or as described herein may also be selected from the group consisting of:
  • the puromycin derivatives of the present invention or the puromycin derivatives to be employed in the methods provided herein are particularly advantageous for the production of alloproteins, e.g. the alloproteins as defined herein.
  • said puromycin derivatives are especially suitable for the production of alloproteins by conjunction, particularly C-terminal conjugation, of certain compounds to proteins, peptides or polypeptides.
  • Said compounds to be conjuncted with said proteins, peptides or polypeptides may be, but are not limited to, markers or labels, like (oligo-)nucleotides, like, e.g.
  • DNA- or RNA- probes reactive groups for affinity labelling, crosslinking or attachment to macromolecular or solid surfaces and vesicles, like e.g. (oligo-)saccharides, affinity ligands like e.g. lipids, spectroscopic labels for UV/VIS- and fluorescence-spectroscopies including single molecule and fluorescence energy transfer spectroscopy, spin labels and metals, metal complexes and metal clusters.
  • puromycin derivatives of the present invention or the puromycin-derivatives to be employed in the methods provided herein are particularly advantageous for the immobilisation of, e.g., proteins, peptides or polypeptides onto certain surfaces, like, e.g., glas surfaces, sepharose surfaces or polymeric surfaces, like, e.g. polystyrene surfaces. Said surfaces may be covered by further substances, like, e.g. streptavidin, biotin or polylysine.
  • puromycin derivatives carrying an azide group e.g. the puromycin derivative indicated under (i), above, are particularly preferred.
  • the cell-free translation system described herein and to be employed in the context of this invention is a cell-free translation system, wherein said release factor contained and to be specifically inactivated by precipitation and/or crosslinking, whereas all other components remain intact, is of prokaryotic or eukaryotic origin, more preferable it is of prokaryotic origin, even more preferably it is from E.coli.
  • a release factor to be inactivated from E. coli may be the release factor 1 , the release factor 2, the release factor homolog 1 , the release factor homolog 2, the release factor homolog 3 or the release factor homolog 4.
  • Said release factors may be encoded by the nucleotide sequences as shown in SEQ ID NOs: 5, 7, 9, 11 or 13 and/or may have the amino acid sequences as shown in SEQ ID NOs: 6, 8, 10, 12, 14 or 37.
  • said release factor contained in said cell-free translation system and to be incredated is the release factor 1 from E. coli.
  • Said most preferred release factor may be encoded by the nucleotide sequence as shown in SEQ ID NO: 5 and/or may have the amino acid sequence as shown in SEQ ID NO: 6.
  • the release factor contained and to be inactivated in the cell-free translation system of the present invention may be different from the release factor, against which the antibody to be employed was directed and/or generated.
  • the release factor contained and to be inactivated in the cell-free translation system of the present invention may be from E. coli.
  • sad translation system comprises RF1 from E. coli.
  • the anti-release factor antibody, precipitating and/or crosslinking said release factor was generated against a release factor from Thermus thermophilus, namely against RF1 from Thermus thermophilus.
  • Said RF1 from Thermus thermophilus may be encoded by the nucleotide sequence as shown in SEQ ID NO: 1 and/or may have the amino acid sequence as shown in SEQ ID NO: 2.
  • the release factor to be inactivated by a specific crosslinking and/or precipitating antibody may be from rabbit, fruit fly or yeast.
  • said release factor to be inactivated by antibodies is a rabbit RF.
  • said release factor is the release factor 1 or the release factor 3 from rabbit, release factor 1 from fruit fly or the release factor 1 or the peptide chain release factor 1 from yeast.
  • Said exemplified release factors may be encoded by the nucleotide sequences as shown in SEQ ID NOs: 15, 17, 19 or 21 , respectively, and/or may have the corresponding amino acid sequences as shown in SEQ ID NOs: 16, 18, 20 or 22, respectively.
  • Corresponding antibodies may be prepared by methods known in the art, for example by the generation of a polyclonal serum against said release factors.
  • "inactivating antibodies to be employed in the cell-free translation system of the present invention are, as described herein, antibodies and/or antibody molecules which are capable of precipitating and or crosslinking the release factor(s) comprised in the cell-free translation system of the present invention.
  • Said “inactivation” may be a complete or a partial inactivation.
  • said "inactivation” leads to an inactivation of the function of said release-factors of at least 60%, more preferably of at least 70%, more preferably of at least 80% and more preferably of at least 90%.
  • the corresponding inactivation of the release-factors by the addition of the precipitating and/or croslinking antibodies and/or antibody molecules can be measured by methods known in the art, as already mentioned above.
  • the release factor contained in said cell-free translation system and to be inactivated may be selected from the group consisting of:
  • a release factor encoded by a nucleotide sequence comprising a nucleotide sequence as shown in any one of SEQ ID NOS: 1; 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21, 23, 25, 27, 29, 31 , 33 and 35;
  • a release factor which is encoded by a nucleotide sequence of a nucleic acid molecule that hybridizes to the complement strand of a nucleic acid molecule comprising a nucleotide sequence as defined in (a) or (b) and which releases a translation product from a ribosome in a cell-free translation system;
  • a release factor which comprises an amino acid sequence as shown in any one of SEQ ID NOS: 2; 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 and 37;
  • a release factor which comprises an amino acid sequence which is at least 40% identical to the full length amino acid sequence as shown in any one of SEQ ID NOS: 2; 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 and 37; and
  • hybridizes refers to hybridization under conventional hybridization conditions, preferably under stringent conditions, as for instance described in Sambrook and Russell (2001), Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor, NY, USA.
  • Polynucleotides which hybridize to the complement strand of a nucleic acid molecule, comprising a nucleotide sequence as defined herein, can, in principle, encode a polypeptide having release factor activity from any organism expressing such polypeptides or can encode modified versions thereof.
  • Polynucleotides which hybridize with the polynucleotides as defined in connection with the invention can for instance be isolated from genomic libraries or cDNA libraries of bacteria, fungi, plants or animals.
  • such polynucleotides are of procaryotic origin, particularly preferred from Thermus thermophil ⁇ s or E. coli.
  • the release factor contained in said cell-free translation system may also be least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, even more preferably at least 90% and most preferably at least 95% identical to the full length amino acid sequences as shown in any one of SEQ ID NO: 2; 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 and 37.
  • the cell-free translation system of the present invention is a cell-free translation system, wherein said precipitating and/or crosslinking anti-RF antibody is directed against a release factor as defined herein-above.
  • a crosslinking and/or precipitating antibody against the RF1 from Thermus thermophilus is employed in particular in cell-free translation/transcription systems of prokaryotic origin (i.e. in E. coli cell- free systems).
  • Said anti-RF antibody may also be monoclonal or polyclonal, preferably polyclonal.
  • Said anti-RF antibody may be purified.
  • said anti-RF antibody is provided in a serum.
  • said cell-free translation system is an E. coli cell-free translation system
  • said anti-RF antibody precipitating and/or crosslinking a release factor contained in said cell-free translation system is an antibody directed against the release factor 1 from Thermus thermophilus.
  • the release factor contained and to be inactivated in said cell-free translation system is release factor 1 (RF1) from E. coli.
  • antibodies and/or polyclonal sera directed against RF1 of Thermus thermophilus are to be employed, in a preferred embodiment of the invention, in E. coli derived translation systems, also antibodies and/or sera and/or antibody molecules specifically directed against E. coli RF1 may be employed.
  • Antibodies against RF1 from E. coli possess the same RF1 deactivation properties as a precipitating and/or crosslinking antibody against RF1 of Thermus thermophilus .
  • the precipitating and/or crosslinking sera specific for RF1 of E. coli and generated against RF1 of E. coli have to be added at higher concentration.
  • the reason may be that the amount of anti- RF antibody molecules, obtained by immunization of a non- human vertebrate, is lower in serum obtained by immunization with RF1 from E. coli than in serum obtained by immunization with RF1 from Thermus thermophilus.
  • the dilution of the translation mixture caused by increasing amount of sera and increasing amount of contaminating material may be the cause of translation inhibition.
  • This in some cases, may restrain the use of "anti-E. coli RF" protein antiserum in translation systems derived from E. coli.
  • the use of an antibody and/or serum against RF1 from Thermus thermophilus is more preferred, in particular in translation systems derived from E. coli.
  • the same amount of added antibodies result in approximately the same incorporation of puromycin derivative.
  • immunization of rabbits with the T. thermophilus RF1 may lead to generation of higher titer of antibodies.
  • environmental temperature may be important in the generation of valuable antibodies against RFs, preferably antibodies against RF1.
  • the protein from termophilic organism living at 65°C should be conformationally "frozen” in rabbit blood (37°C) and therefore be more homogenic matrix for antibody formation.
  • E. coli protein is under same conditions as in nature and can possess conformation flexibility.
  • the present invention relates to a kit comprising the cell free translation system of the present invention.
  • said kit comprises individual ingredients of the cell-free system and in particular the two essential further components of the inventive cell-free systems, namely an anti-RF antibody to be added to the cell-free translationsystem and nonsense-codon suppressing agent(s).
  • the cell- free translation system, the antibody (antibody preparation and/or serum) and the nonsense-codon suppressing agent(s) are individually packed in said kit, preferably in individual vials.
  • all components of the invention are already comprised in one vial.
  • a particular preferred antibody and/or serum to be used in the cell-free translation system of the present invention is an antibody and/or serum directed and or generated against RF1 of Thermus thermophilus.
  • Said antibody and/or serum is particulary preferred in prokaryotic cell-free translation systems, e.g. translation systems derived from E. coli. It was surprisingly found, that generated against RF1 of Thermus thermophilus are capable of efficiently inhibiting and/or inactivating and/or deactivating the function of E. coli RF1. Accordingly, the present invention also relates to an anti-RF antibody obtainable by
  • anti-RF antibody in particular "anti-RF1 antibody” as employed in context of the present invention refers to an antibody, a plurality of antibodies and/or a serum comprising such antibodies which is/are able to specifically bind to, interact with, detect and/or precipitate and/or crosslink RFs, in particular RF1 , preferably RF1 from E. coli or a fragment thereof. Said term also relates to a purified serum, i.e. a purified polyclonal serum.
  • the antibody molecule is preferably a full immunoglobulin, like an IgG, IgA, IgM, IgD, IgE, IgY (for example in yolk derived antibodies).
  • antibody as used in this context of this invention also relates to a mixture of individual immunoglobulins. Furthermore, it is envisaged that the antibody/antibody molecule is a fragment of an antibody, like an F(ab), F(abc), Fv Fab' or F(ab)2. Furthermore, the term “antibody” as employed in the invention also relates to derivatives of the antibodies which display the same specificity as the described antibodies. Such derivatives may, inter alia, comprise chimeric antibodies or single- chain constructs. Yet, most preferably, said "anti-RF(1) antibody” relates to a serum, more preferably a purified (polyclonal) serum and most preferably to a crude polyclonal serum.
  • the antibody/serum is obtainable, and preferably obtained, by the method described herein and illustrated in the appended examples.
  • the term "eliciting an in vivo humoral response in a non-human vertebrate” relates to the provocation of an immune response in a non-human vertebrate, in particular the provocation of an antibody response to RF1 from Thermus thermophilus or a fragment thereof.
  • Said antibody response comprises primary as well as secondary antibody responses to the antigenic challenge with RF1 from Thermus thermophilus or a fragment thereof.
  • the term “eliciting an in vivo humoral response” accordingly, relates to the provocation of an immune reaction involving the production of antibodies directed towards the antigen, namely RF1 from Thermus thermophilus or a fragment thereof.
  • Said release factor 1 from Thermus thermophilus or said fragment thereof may be naturally occurring release factor 1 or it may, preferably, be recombinantly produced.
  • Naturally release factor 1 may be purified by methods known in the art and also the recombinantly produced RF1 may be further purified before a non-human vertebrate is immunisized with said RF1 or an RF1 comprising preparation.
  • said release factor 1 from Thermus thermophilus or said fragment thereof may be purified.
  • the term "RF 1 from Thermus thermophilus or a said fragment thereof is purified” relates preferably to an isolated RF1 from Thermus thermophilus protein or fragment thereof, which has been purified to homogeneity.
  • the coding sequence and/or the protein sequence of RF1 of Thermus thermophilus is known in the art; see also SEQ ID No. 1 and 2. In particular, it has been purified to a purity level of at least 80%, more preferably of at least 85%, even more preferably of at least 90%, particularly preferred of at least 95% purity.
  • the purity of RF1 from Thermus thermophilus protein may be confirmed by methods known in the art.
  • the purified preparation of RF1 from Thermus thermophilus to be employed in the immunization protocols described herein comprises, preferably, less than 5% contaminating, unrelated proteins or protein fragments. Most preferably, said preparation comprises less than 2% contaminating, unrelated proteins or protein fragments.
  • Purity of the purified RF1- preparation may be measured by methods known in the art which comprise gel stainings (in particular silver stains of SDS-PAGE followed by densitometric analysis) NMR-measurements or mass spectroscopy (MS), in particular, MALDI mass spectroscopy.
  • said RF1 from Thermus thermophilus or a fragment thereof may be purified by ion exchange chromatography.
  • Said purification may further comprise a gel filtration.
  • Said purification may further comprise, prior to ion exchange chromatography, a protein precipitation step, preferably an AMS-precipitation step.
  • Said purification may further comprise, after the ion exchange chromatography a hydroxyapatite chromatography step and/or an additional precipitation step and/or additional ion exchange chromatography step and/or additional ammonium-sulfate-precipitation step. More preferred, said purification may further comprise, most preferred after the first ion exchange chromatography, a heat treatment. Said heat treatment is preferred. For example and more preferred, said heat treatment may be for 15 minutes at 65 °C.
  • Ion exchange chromatography is known to the artisan and ion exchange media comprise, but are not limited to Mini beads Q, Source 15 Q, Source 30 Q, Sepharose High Performance Q, Sepharose Fast Flow Q 1 Sepharose XL Q, Sepharose Big Beads Q, DEAE, Streamline DEAE (all from Amersham Biosciences, Vienna, Austria), DEAE-cellulose, QA-cellulose, CM-cellulose, SE-cellulose, DE-52 (Whatman, Kent, England) or Agarose based ion exchangers. Most preferably a Q- Sepharose FF column (Amersham Biosciences, Vienna, Austria) or an EMD-SO3 column (Merck) is employed. It is of note that also normal gravity flow or FPLC systems may be employed.
  • Gel filtration systems and media are also known to the skilled artisan which comprise Superdex peptide, Superdex 30, Superdex 200, Superose 6, Superose 12, Sephacryl, Sphadex, Biogel P, Agarose-gel, Fracto-gel or Ultro-gel.
  • Protein precipitation techniques comprise, inter alia, Dextran sulphate-, Polyethylene glycol (PEG) 4000 - 8000-, Acetone-, Protamne sulphate-, Streptomycin sulphate-, pH-shift-precipitations.
  • said protein precipitation is carried out by ammonium sulfate precipitation as known in the art.
  • a preferred anti-RF antibody of the present invention as obtained in the eliciting step (a) of the above recited method may be purified or may be provided in a serum.
  • Said non-human vertebrate, from which said serum containing said anti-RF antibody and/or said anti-RF antibody is obtained, may be selected from the group consisting of rat, mouse, rabbit, chicken, sheep, horse, goat, pig and donkey.
  • said non-human vertebrate is rabbit.
  • the anti-RF antibody of the present invention may also obtainable by eliciting an in vivo humoral response against a native release factor from Thermus thermophilus or a fragment thereof in a non-human vertebrate.
  • the (purified) RF1 from Thermus thermophilus may be a native, RF1 from Thermus thermophilus as defined herein. It is preferred that said RF1 from Thermus thermophilus is a full length protein, comprising preferably 354 amino acids.
  • the antibodies of the present invention provide for the first time a reliable tool to (exhaustively) precipitate and/or crosslink RFs and thereby (exhaustively) deplete said RF's from e.g. cell-free translation systems.
  • the anti-RF1 antibodies produced according to the above-described method are, in contrast to e.g. aptamers against RFs, capable of reliably depleting RFs, in particular RF1 from E. coli from cell-free translation systems.
  • the present invention also relates to the use of an anti-RF antibody as defined herein-above and/or the anti-RF antibody and/or serum of the present invention for the preparation of a cell-free translation system.
  • a method for the production of an anti-RF antibody comprising the steps of
  • said release factor 1 from Thermus thermophilus or said fragment thereof may be purified.
  • said anti-RF antibody as obtained in the eliciting-step (a) may be purified.
  • said release factor 1 from Thermus thermophilus or said fragment thereof is a native-release factor from Thermus thermophilus or a fragment thereof.
  • the RF1 from Thermus thermophilus has the amino acid sequence as shown in SEQ ID No. 2 or is encoded by a nucleic acid molecule as shown in SEQ ID No. 1.
  • the term "RF1 of Thermus thermophilus” also comprises variants and derivatives of said RF1 from Thermus thermophilus.
  • said variants and derivatives comprise at least 80% identity in amino acid sequence to the Thermus thermophilus-RFI sequence as shown in SEQ ID No. 2.
  • Said fragments preferably, comprise at least 10, more preferably at least 12, more preferably at least 15 amino acid residues.
  • said fragments may be linked to bulk proteins, like KHL in order to facilitate the immunization in non-human vertebrates.
  • the present invention also relates to a method for the production of alloproteins, comprising the step of translating RNA into translation product in a cell-free translation system as disclosed herein.
  • alloproteins refers to proteins that are achieved by applying the subject-matter of the present invention. Said term also refers to proteins having covalently bound a non-proteinaceous molecule which usually is not part of (the )naturally occurring protein(s). Said alloprotein may, for example, comprise a puromycin and/or derivative thereof as defined herein. Furthermore, said proteinaceous molecule may comprise an unnatural amino acid (for examples see, Gilmore (1999), Topics in Current Chemistry, 202, 77-99). Furthermore, said molecule being covalently bound to and comprised in the alloprotein, might be a functional substituent. Various functional substituents of proteins are well-known in the art.
  • these functional substituents may be oligosaccharides, lipids, fatty acids, phosphates, acetates or other functional groups to modify polypeptide chains of functional proteins (e. g. Eisele (1999), Bioorganic and Medicinal Chemistry 7,193-224).
  • said molecule might be a residue of a puromycin (-derivative) as defined herein and/or a puromycin (derivative) as defined herein itself and/or the puromycin derivative of the present invention itself.
  • the alloproteins produced by the method of the present invention may be used in a wide variety of applications, for example the preparation of synthetic enzymes (Corey (1987), Science, 238, 1401-1403), gene therapy (Zanta (1999), Proc. Natl. Acad. Sci. U.S.A., 96, 91-96), construction of protein microarray (Niemeyer (1994), Nucleic Acid Res., 22, 5530-5539), creation of molecular scale devices (Keren (2002), Science, 297, 72-75), and development of immunological assays (Niemeyer (2003), Nucleic Acids Res., 31, e90).
  • the method for the production of alloproteins offers the possibility that any desired chemical structure including different dyes, affinity tags, spin labels etc. may be covalently conjugated with proteins at a high yield.
  • puromycin modified with an azide group may be used to covalently attach to the C-terminus of proteins for subsequent one site addressed Staudinger reaction (Kohn (2004), Angew. Chem. Int. Ed Engl., 43, 3106-3116) and utilization of puromycin carrying ⁇ -thio-ester group may allow to use the protein ligation technology (Lovrinovic (2003), Chem. Commun. (Camb. ), 822-823).
  • This conjugation technology can further be used to develop concepts for preparation of protein arrays and novel tools to study protein interactions (Ramachandran (2004), Science, 305, 86-90).
  • puromycin derivatives modified with an azide group may also by used for site-specific protein immobilisation, e.g. by taking advantage of the Staudinger ligation (Soellner, 2003, loc. cit.).
  • said alloproteins may comprise, as proteinaceous part, proteins selected from the group consisting of enzymes, growth factors, cytokines, toxins, hormones, pheromones, structural proteins and the like. It is also envisaged that said alloproteins only comprise fragments, like functional, active fragments of said enzymes, hormones, pheromones, growth factors, cytokines, toxins, structural proteins and the like. Said proteins may act as act as core-proteins and/or starting proteins for the alloproteins to be produced in the cell-free translation system of the present invention and corresponding additional chemical structures may be added to said proteinaceous part.
  • Said alloproteins may also be conjugates of proteins and nucleic acids, preferably nucleic acids having (a) specific sequence(s). Said conjugates allow to link the properties of these two distinct groups of biopolymers within one molecule. Therefore, said conjugates can be used in a wide variety of applications, where said linkage of said properties of these two distinct groups of biopolymers is advantageous.
  • Applications of alloproteins as described herein are well known in the art and may, for instance, include the preparation of synthetic enzymes (Corey (1987), Science, 238, 1401-1403), gene therapy (Zanta (1999), Proc. Natl. Acad. Sci.
  • the alloproteins or product produced by the method of the present invention may comprise esterases.
  • esterases refers to a molecule which catalyzes the cleavage of an ester into an alcohol and an carboxylic acid.
  • alcohol refers to a compound carrying at least one hydroxyl group and the term “carboxylic acid” refers to a compound carrying at least one carboxyl group.
  • said alloproteins may comprise the esterase 2 from Alicyclobacillus acidocaldarius (Manco, G. (1998), Biochem.
  • a growth factor (or any other protein) may be produced which comprises at least, e.g. one additional unnatural amino acid or (e.g.) a puromycin-derivative as defined herein.
  • the Esterase (Est2) from Alicyclobacillus acidocaldarius (SEQ ID NO: 39 or 55) is a thermostable enzyme that consists of one polypeptide chain and possesses a broad substrate specificity (Manco, G. (1998) Biochem. J 332 ( Pt 1), 203-212). Due to high thermostability, practically instant folding and refolding and easily detectable activity, this esterase has a potential application as a reporter for in vitro and in vivo protein expression systems. The tertiary structure of the esterase was determined by X-ray crystallography (De Simone, G. (2000) J MoI. Biol 303, 761-771).
  • Serine 155 located in the Ser-His-Asp catalytic triad (Fig. 16B.), is essential for hydrolytic activity (De Simone, G. (2000) J MoI. Biol 303, 761-771) It is encoded by the ACG triplet at the corresponding position of the est2 mRNA (Hemila (1994), Biochim. Biophys. Acta 1210, 249-253). As already mentioned before and exemplified herein below, the coding sequence for serine 155 was substituted to a RF1 -dependent stop codon (UAG) and the resulting construct was used to test the conditions for efficient termination and/or suppression at UAG stop codon.
  • UAG RF1 -dependent stop codon
  • Est2 mRNA(amber 155) from the template pEst2_amber 155 (see herein below) allows to monitor in parallel the efficiency of the UAG suppression by a band shift in SDS- PAGE and the accumulation of esterase activity in the in vitro translation mixture. This assay is suitable for estimation of optimal conditions to achieve highly efficient suppression in different in vitro translation mixtures. Such assessment seems to be very important since translation systems may individually differ from each other due to different source and preparation method.
  • said alloproteins are proteins that have covalently attached puromycins or and/a derivative thereof. Furthermore, said alloproteins may have incorporated an (unnatural) amino acid delivered by suppressor aminoacyl-tRNA. Said suppressor aminoacyl-tRNA may be e.g. suppressor serine-tRNA Ser(CVA) Corresponding additional residues, covalently attached to said puromycin or said derivative thereof, have been described herein above. Also the linker between the puromycin (-derivative) and said residue have been described above and are employed in the context here, mutatis mutantis. Most preferably in the herein provided method for the production of alloproteins, said alloproteins are proteins which have, at their C-terminus a puromycin or a corresponding derivative covalently attached.
  • the present invention also provides for synthetic puromycin derivatives which are particulary useful in context of the preparation of alloproteins.
  • Said alloproteins are preferably prepared using the method disclosed herein and employing the cell-free translation system of the present invention.
  • the present invention also provides for a puromycin derivative having a residue covalently attached directly or via a linker to one of its elements selected from the group consisting of:
  • the puromycin derivatives described herein are to be employed in the generation of alloproteins. Said derivatives may be selected from the group consisting of: 44
  • the puromycin derivative of the present invention or the puromycin-derivative to be employed in methods provided herein may be particularly useful in the cell-free translation system, the kit comprising said cell-free translation system, the method for the production of alloproteins.
  • the puromycin derivative of the present invention may be useful in mRNA display.
  • the yields of the mRNA-protein coupling in mRNA display Robots, (1997) JW Proc Natl. Acad. Sci. U.S.A. 94, 122297-302) are usually low. The reason is the low tolerance of the ribosomal A-site for 5'-extended puromycin-nucleic acid conjugates and the high selectivity of this site for EF-Tu.
  • RNA molecules that are longer than 5-6 nucleoitde residues can not enter the ribosomal A- site in EF-Tu. GTP independent manner (Fig. 11).
  • puromycin derivative of the present invention instead of covalent attachment of RNA to the 5'-position of puromycin an alternative strategy by which the RNA (mRNA) or other functional groups are attached directly or via a linker to the nculeobases of puromycine-derived olignucleotides (e.g. CpCpPu or CpPu) can be used.
  • a linker to the nculeobases of puromycine-derived olignucleotides e.g. CpCpPu or CpPu
  • the present invention also provides for the use of a puromycin derivative having a residue covalently attached directly or via a linker to one of its elements selected from the group consisting of:
  • the residue is selected from the group consisting of a Cy3-fluorosphore, biotin or another affinity tag, a reactive group for affinity labelling or any other reporter group. Further, the residue may be a(n) (other) spectroscopic reporter.
  • the linker is an aliphatic amine derivative, in particular an aliphatic amine forming an amide with a fatty acid as described above.
  • the puromycin derivative disclosed herein may be selected from the group consisting of:
  • A Biosynthesis of proteins in all living organisms is based on peptidyl transferase reaction catalyzed by the ribosome. A nucleophilic attack on the carbonyl group of the ester bond of the peptidyl-tRNA by the amino group of the aminoacyl-tRNA results in formation of an amide (peptide) bond.
  • the antibiotic puromycin that by its structure resembles the aminoacylated 3'-terminal adenosine of the aminoacyl-tRNA, can compete with aminoacyl-tRNA.EF-Tu.GTP ternary complex for the ribosomal A-site. In such case it serves as a low molecular weight acceptor substrate in peptidyl transferase reaction leading to formation of puromycin-peptide that is released from ribosomes (Pavlov (1998), J MoI. Biol 284, 579-590).
  • Nucleotide sequence of plVEX 2.3d-Est2 (pEst2; SEQ ID NO: 40).
  • Bolded letters are the coding sequence (SEQ ID NO: 38) of esterase 2 (SEQ ID NO: 39 or 55).
  • Puromycin derivatives used for the synthesis of conjugates with esterase 2 within the present application or for incorporation into other proteins, peptide or polypeptides to produce alloproteins.
  • the puromycin derivatives were radioactively labelled or can be radioactively labelled as described herein (Example 8).
  • R oligonucleotide, affinity label, spectroscopic label, biotin, Cy3, azide group, hydrogen residue or other residue, either with or without an additional linker.
  • Alternative positions for attachment of functional groups directly or via a linker are indicated by numbers (5, 6, 8).
  • A Radioactive image of SDS-PAGE of 2 ⁇ L samples withdrawn from translation mixture after 90 min. of incubation in the presence of 6 ⁇ M [ 32 P]GpCpPuromycin and different amounts of antibodies against RF1 from Thermus thermophilus, Lane 1 , no antibodies added. In lanes 2, 3, 4, 5, 6, 7, 8, 9 the rabbit serum anti-RF1 antibodies were diluted 1 :3000, 1 :300, 1 :150, 1:75, 1:40, 1 :30, 1 :15 and 1:7.5, respectively. Position of the full-length esterase is indicated by arrow.
  • B The dependence of the reaction yield upon added amount of anti-RF1 antibodies.
  • the reaction yield was determined as a ratio between pmols of [ 32 P]GpCpPuromycin incorporated into the full-length esterase and pmols of the full-length synthesized esterase (the initial values were calculated from corresponding bands on radioactive images).
  • Lane 1 no puromycin derivative added
  • Lane 2 1 ⁇ M [32P]p-CC-puromycin (42 ⁇ M; 301 mCi/pmol)
  • Lane 3 5 ⁇ M [32P]p-CC-puromycin (42 ⁇ M; 301 mCi/pmol)
  • Lane 4 10 ⁇ M [32P]p-CC-puromycin (42 ⁇ M; 301 mCi/pmol)
  • the arrows indicate the position of the esterase.
  • RNA 50 nucleotide long RNA Oligonucleotides with strongest affinity to RF1 from E. coli (see, Szkaradkiewicz (2002), FEBS Lett. 514, 90-95)
  • RNA 50 nucleotide long RNA Oligonucleotides with strongest affinity to RF1 from E. coli (see, Szkaradkiewicz (2002), FEBS Lett. 514, 90-95)
  • CC-Puromycin in the cell-free translation system programmed by pEst2.
  • 2.5 ⁇ M [32P]p-CC-puromycin (301 mCi/pmol) was used for in-vitro translation together with 500 nM PCR produced DNA fragments containing sequence coding for RNA Aptamer, 2 nM pEst2.
  • Lane 1 no aptamer
  • Lane 2 presence of aptamer #3
  • Lane 3 Presence of aptamer
  • CC-puromycin (301 mCi/pmol) was used for in-vitro translation together with
  • the arrow indicates the position of the full-length esterase. Incubation time intervals are indicated above corresponding lanes.
  • Kd was approximatively 50 nM (see, Szkaradkiewicz (2002), FEBS Lett. 514, 90-95).
  • Biotin-[ 32 P]pCpPuromycin was added; lane 4, Cy3-[ 32 P]pCpPuromycin was added.
  • Position of the full-length esterase is indicated by an arrow.
  • Radioactive image of the SDS-gel, lane 1 : control (without puromycin derivative), lane 2, 3, 4, 5, 6 are the images of reaction mixtures after incorporation of 2, 4, 7, 9 and 11 nucleotide long deoxyribonucleotides attached to the puromycin (for examples see Fig. 4 and Fig 23), respectively.
  • Translation assay contained 11 ⁇ M puromycin-modified deoxyoligonucleotides. In lane 2, 0.5 ⁇ l of translation mixture were loaded in order to decrease the signal from truncated products.
  • Translation assay contained 31 ⁇ M puromycin-modified deoxyoligonucleotides.
  • Translation assay contained 94 ⁇ M puromycin-modified deoxyoligonucleotides.
  • A Enzymatic activities of the esterase2 conjugated with GpCpPuromycin (control) and of esterase2 having incorporated GpCpPuromycin carrying a biotin on the N 4 of the 5'-cytosin residue and being immobilized to streptavidin-coated polystyrene plates.
  • FIG. 13 Immobilization of Esterase-Puromycin conjugates produced on streptavidin coated glass surface. Translation reaction was performed as described in Experimental section. Plate 1 is a control onto which 1 ⁇ L purified esterase, prepared by purification from E. coli cells overexpressing the enzyme according to (Arkov (2002), J Bacteriol. 184, 5052-5057). On the plates 2, 3, and 4 one ⁇ L translation mixture containing no puromycin derivative (2), Biotin-CpPuromycin (3) and Biotin-CpPuromycin (4) together with anti-RF1 antibodies, respectively. After 90 min. at 37°C the plates were rinsed by tap-water and the esterase activity was determined by 2-naphthyl acetate assay and developed with Fast Blue BB salt.
  • Nucleotide sequence of pEst2_amb155 (SEQ ID NO: 41). Bolded letters are the coding sequence of esterase 2 Ser155amber mutant.
  • A Kinetics of in vitro transcription/translation of the pEst2_Amb155; filled triangles: pEst2 (control) as a template; filled squares: pEst2_Amb155 as a template.
  • the concentration of the newly synthesized proteins was determined by measurement of the TCA precipitatable [ 14 C]leucine radioactivity in aliquots withdrawn at indicated time intervals.
  • B Radioactive image of the SDS-PAGE of 2 ⁇ l samples from the in vitro translation mixture after 120 min. incubation time. Lane 1, pEst2_Amb155 as a template; lane 2, pEst2 (control) as a template.
  • Serine 155 is a residue assential for catalysis.
  • A Autoradiography of 14 C-labelled in vitro translation products by SDS PAGE of translated products: lines 1 , 2 and 3 amber UAG 155 as a template, line 4 is a control with Ser 155 codon as an template.
  • Line 1 complete translation mixture (polypetide terminated at position 155), line 2; RF1 depleted by RF1 antibodies (nearly cognate aminoacyl-tRNAs suppress inefficiently the amber code, since essential serine is missing, no activity ), line 3; RF1 depleted by RF1 antibodies and amber suppressor tRNA Ser added (efficient synthesis of active, serine -155, esterase).
  • B Ezymatic activity of translated products measured by 4-nitrophenol assay. Activity bars correspond to SDS PAGE lanes.
  • A Translation was carried out in the absence of anti-RF1 antibodoes. Radioactive image of the gel after SDS-PAGE separation of 2 ⁇ l samples from the in vitro translation mixture is presented. Concentration of added suppressor tRNA Ser(CUA) was as follows: lane 1 , no tRNA Ser added, lane 2, 24 nM, lane 3, 120 nM, lane 4, 600 nM, lane 5, 2.5 ⁇ M, lane 6, 10 ⁇ M, lane 7, 25 ⁇ M tRNA Ser added.
  • B Translation was carried out in the presence of anti-RF1 antibodies. Radioactive image of the gel after SDS-PAGE separation of 2 ⁇ l samples from the in vitro translation mixture is presented. Concentration of added suppressor tRNA Ser was the same as described in (A).
  • SPOT In vitro protein selection
  • reaction yield was determined as X/Y*100 where X are pmols of
  • A Activity of the esterase conjugated with GpC(Biotin)pPuromycin immobilized to Streptavidin-Sepharose.
  • In vitro transcription/translation was carried out as described in experimental section (Example 20) in the presence of: lane 1 : no antibodies, no puromycin derivative; lane 2: no antibodies, 7 ⁇ M of C(Biotin)pPuromycin; lane 3:2 ⁇ l of anti RF1 serum per 30 ⁇ l of reaction mixture, no puromycin derivative; lane 4: 2 ⁇ l of anti RF1 serum per 30 ⁇ l of reaction mixture, 7 ⁇ M of C(linker-NH2)pPuromycin; lane 5: 2 ⁇ l of anti RF1 serum per 30 ⁇ l of reaction mixture, 7 ⁇ M of C(Biotin)pPuromycin;
  • Examples of derivatives of CpPuromycin modified on the 5 ⁇ -end of the cytidine residue can be in the shown radioactive labelled form or in the corresponding non-radioactive labelled form.
  • Degenerated primers were used to amplify a prfA specific probe from Thermus thermophilus genomic DNA by PCR. Preparation of T. thermophilus genomic DNA and subsequent genomic PCR followed conventional protocols for mesophilic bacteria (Sambrook (2001), Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, NY, Vol. 1, 2, 3). A 50 mg bacterial pellet in an Eppendorf tube was resuspended in 565 ⁇ l TE buffer, 30 ⁇ l 10% SDS and 5 ⁇ l 20 mg/ml Proteinase K was added and incubated for 1 h at 37° C.
  • Lysis was performed after addition of 100 ⁇ l 5 M NaCI and repeated uptaking and emptying the bacteria with a needle-equipped syringe by shearing forces. After addition of 80 ⁇ l of 10% Hexadecyltrimethylammoniumbromid (CTAB) in 0.7 M NaCI, 10 min incubation at 65° C and extraction with 700 ⁇ l Chloroform/lsoamylalkohol and 5 times with 700 ⁇ l Phenol/Chloroform/lsoamylalcolhol 25:24:1, genomic DNA was precipitated with Isoamylalcohol.
  • CTAB Hexadecyltrimethylammoniumbromid
  • 1 ⁇ g DNA from the genomic DNA preparation was used in a 100 ⁇ l PCR reaction containing 5 ⁇ l of each 10 ⁇ M primer, 10 ⁇ l 15 mM MgCI__2 , 10 ⁇ l 10x Taq Pol buffer (10OmM Tris-HCI pH 8.8, 500 mM KCI, 15 mM MgCI 2 ), 1 ⁇ l 1 U/ ⁇ l Taq Polymerase, and cycled 30 times with 30 s at 95°, 30 s at 60° and 60 s at 72° after an initial 5 min denaturation at 95°.
  • a 3.0 kbp fragment could be identified carrying the complete prfA sequence.
  • the fragment was cloned into the plasmid pBluescript KS+ and the resulting plasmid pBlueK4b was sequenced.
  • the sequence identified is identical to that of the literature (Ito (1997), Biochimie. 79, 287-292) and is shown in SEQ ID NO: 1.
  • the corresponding amino acid sequence is shown in SEQ ID NO: 2.
  • Example 2 Overexpression of the release factor 1 of Thermus thermophilus in E. coli and purification of the same
  • the T.th.RF1 protein was heterologous overexpressed in E.coli and purified to homogeneity as judged by SDS PAGE and Maldi mass spectroscopy.
  • S 100 supernatant from the ultracentrifugation was concentrated by AMS- precipitation, dialyzed against 50 mM Tris/HCI pH 7.5, 10 mM MgCI 2 , 1 mM ⁇ - mercaptoethanol, 5% glycerol and used for Q-Sepharose FF (Amersham-Pharmacia) ionexchange chromatography running a gradient from 0 to 500 mM NaCI.
  • RF1- containing fractions were pooled, 15 min at 65°C heat-treated removing most E.coli proteins - including heterologous E.coli RF1 - AMS-precipitated, dialyzed against 10 mM K-Phosphate buffer pH 6.8 and used for hydroxyapatite chromatography (Merck) running a gradient from 10 to 500 mM K-Phosphate pH 6.8.
  • Thermus thermophilus RF1 was used to immunize two rabbits following the standard one month immunization- protocol at Eurogentec (Seraing, Belgium): A first immunization used the glycerinated protein mixed with incomplete Freund's adjuvant and a intradermic multisite injection at the rabbits back at day 0. Three boost immunizations at day 14, 30 and 60 followed with a small bleeding after 45 days and termination of the rabbits and final bleeding after 70 days. Vacutainer tubes were used to process blood samples and remove agglutinated blood clots.
  • Wild type E.coli RF1 cloned in pET11a was used to subclone the protein into pET28, adding a N-terminal (His) 6 -tag. Overexpression in E.coli gave soluble RF1-(His) 6 used to purify the protein.
  • Antibodies against E. coli RF1 were processed as in Example 3 using the antibody service of Genaxxon Bioscience (Staff Weg, Germany). However, a one-month protocol with immunizations at day 0, 14 and 30 as well as a bleeding at day 45 was followed.
  • Example 6 Preparation of the plasmid plVEX 2.3d-Est2 (pEst2) comprising a cDNA encoding the esterase 2 from Alicyclobacillus acidocaldarius (Est2)
  • est2 gene was amplified by using pT7SCII-esterase as template, recombinant Tag-polymerase, and two synthetic oligonucleotides, estfor (5'- CCATGGCGCTCGATCCCGTCATTCAGC -3'; SEQ ID NO: 42) and estrev (5'- GAGCTCCTAGGCCAGCGCGTCTCG -3'; SEQ ID NO: 43) in a 30-cycle polymerase chain reaction (1 min at 95°C, 30 sec 60°C, and 1 min at
  • the primer estfor was designed to introduce a ⁇ /col restriction site (underlined) at the initiation site which also leads to a C to G exchange (bold) in the coding sequence
  • Primer estrev introduces a Sac ⁇ restriction site (underlined) downstream from the UAG stop codon (bold).
  • the PCR product was eluted from an agarose gel and ligated into pGEM-T vector (Promega) and completely sequenced to verify that only desired mutations were introduced.
  • the obtained plasmid was then digested with ⁇ /col and Sacl, the cloned fragment was eluted from an agarose gel and ligated into ⁇ /col-Sacl-linearized in vitro-translation- vector plVEX 2.3d (Roche Diagnostics, Mannheim, Germany).
  • plVEX 2.3d-Est2 The resulting plasmid, plVEX 2.3d-Est2 (pEst2), was used for in vitro translation.
  • a map of pEst2 is shown in Fig. 2, the corresponding nucleotide sequence is shown in Fig. 3 (SEQ ID NO: 40.
  • the plasmids for coupled in vitro transcription/translation were purified with modified PEG method (Nicoletti (1993), Biotechniques, 14, 532-4, 536). Therefor, a 12.5 ml cell culture, containing the plasmid was harvested and resuspended in 240 ⁇ L of 25 mM Tris/HCI pH 8.0, 50 mM glucose, 10 mM EDTA. Then 600 ⁇ L of 0.2 N NaOH, 1 % (w/v) SDS was added. The tube was gently turned over for several times and incubated for 4 minutes at room temperature.
  • the supernatant was mixed with 300 ⁇ L of isopropanol and incubated for 15 minutes at room temperature. After centrifugation at 16,600 g for 10 min, the DNA pellet was washed two times with 75% ethanol, dried and dissolved in distilled water.
  • the resulting 5'-dimethoxytrityl-protected dinucleotide was cleaved from the CPG matrix by 32% ammonium hydroxide and left under this condition at 65°C for 1h in order to achieve total deprotection. Subsequently the dinucleotide was purified by HPLC and the trityl group was removed by treatment with 80% aqueous acetic acid solution. The final purrification was achieved by two HPLC steps. The sample was concentrated by evaporation and desalted by passing through a SepPac cartridge. Concentration of the puromycin derivatives in the in vitro translation assay was 7 ⁇ M, unless otherwise indicated. Examples for the puromycin derivatives that were used within the present application are shown in Fig. 4, Fig. 5 and 23.
  • the used puromycin derivatives were labelled with [ ⁇ - 32 P]ATP at the 5'-end by 32 P in the following way.
  • the reaction mixture (10 ⁇ l) contains 0.8 U/ ⁇ l T4-polynucleotide kinase, 4 ⁇ M ATP, 0.2 ⁇ M [ ⁇ - 32 P]ATP (10 ⁇ Ci, 4950 mCi/mmol, Hartmann Analytic, Braunschweig, Germany), 4 ⁇ M puromycin-modified oligonucleotide (Purimex, Staufenberg, Germany) in T4-polynucleotide kinase buffer (70 mM Tris/HCI (pH 7.6), 10 mM MgCI 2 , 5 mM DTT). Phosphorylation was carried out for 30 minutes at 37°C.
  • Example 9 The SDS-PAGE (polyacrylamid gel electrophoresis) of the present invention
  • the dried gels were exposed to an imaging plate for radioactivity analysis with the Phosphorlmager SI (Molecular Dynamics, Sunnyvale,
  • Example 10 The esterase activity assays of the present invention
  • Example 11 Depletion of the release factor 1 from E. coli in an E. coli cell-free translation system by precipitating and/or crosslinking said release factor 1 from E. coli with polyclonal antibodies against the release factor 1 of Thermus thermophilus and thereby increasing the incorporation of puromycine and/or its derivatives at the C-terminal nonsense codon of the esterase 2 (Est2) from Alicyclobacillus acidocaldarius
  • kits used for in vitro translation experiments within the present application were evaluation size transcription/translation kits from RiNA GmbH, (Berlin, Germany) and the reaction was performed according to the manual provided by the supplier.
  • [ 14 C]Leucine 25 mCi/mmol, Amersham
  • the template control vector or plVEX 2.3d-Est2
  • the reaction was started by transferring the reaction tube to the thermo shaker at 37 °C with 500 rpm agitation.
  • different amounts of serum were added per 30 ⁇ l of the reaction mixture.
  • Radioactively labeled puromycin derivatives were added up to 6 ⁇ M. Amount of newly synthesized protein was determined from radioactivity of hot 10% trichloroacetic acid precipitate.
  • the reaction mixture should be incubated at 37°C for 2 hours with agitation (500 rpm). Aliquots can be withdrawn at any desired time intervals.
  • the reaction can be performed without radioactivity (Leu should be substituted with the same amount of water and T-mix with Leu (supplied with the Kit) should be used instead of one without.
  • Protocol for in vitro translation (RiNA GmbH Kits) in the presence of anti-RF1 (T. thermophilus) antibodies:
  • reaction mixture should be incubated at 37°C for 2 hours with agitation (500 rpm). Aliquots can be withdrawn at any desired time intervals.
  • reaction mixture should be incubated at 37°C for 2 hours with agitation (500 rpm). Aliquots can be withdrawn at any desired time intervals.
  • the standard in vitro translation system of the present application (without RF depleating agents (e. g. Antibodies against RF1 from Thermus thermophilus) or nonsense codon suppressing agents (e. g. puromycin derivatives and/or suppressor tRNAs)) comprises the following ingredients: - 3OS cell-free extract from E. coli (enzyme- and und ribosomal fraction);
  • RF depleating agents e. g. Antibodies against RF1 from Thermus thermophilus
  • nonsense codon suppressing agents e. g. puromycin derivatives and/or suppressor tRNAs
  • each amino acid can be applied as a 14C amino acid with a concentration of 100 ⁇ M (e.g. 14C-leucine))
  • aprotinin 10 ⁇ g/ml reaction mixture leupeptin 5 ⁇ g/ml reaction mixture, pepstatin 5 ⁇ g/ml reaction mixture; and
  • the E. coli release factor 1 (RF 1) responsible for termination at the UAA and UAG stop codons was inactivated in the in vitro translation system from E. coli by rabbit antibodies specific against Thermus thermophilus RF1.
  • the C-terminal labeling of the esterase was monitored by incorporation of [ 32 P]pGpCpPuromycin into full-length protein (Figure 6A). The activity of the synthesized esterase was also determined ( Figure 6B).
  • oligonucleotides being covalently bound to the 5 " -end of puromycin that are longer than 5-6 nucleotide residues can not enter the ribosomal A-site in EF-Tu. GTP independent manner (Fig. 11).
  • RNA Ribonucleic acid
  • mRNA RNA
  • CpCpPu oligonucleotide
  • Fig. 5 A direct synthesis of puromycin-DNA conjugates can be achieved by synthons that serve as linkers between Cp-Pu and any oligonucleotide synthesised by a standard DNA synthesis.
  • biotin or Cy3 fluorophore were both covalently attached via the exocyclic N 4 of the cytidine residue to 5'[ 32 P]pCpPuromycin (Fig. 4; Fig. 5). These substrates were incorporated with the same efficiency into full-length polypeptide as pGpCpPuromycin (Fig. 10A). The C-terminal incorporation of puromycin derivatives did not lead to any detectable loss of activity of the esterase.
  • Example 12 Immobilization of esterase 2 having incorporated a biotinolated puromycine-derivative on streptavidin coated polystyrene surfaces
  • the streptavidin coated plate treated with C-terminally biotinylated esterase (having incorporated Biotin- pGpCpPuromycin) was enzymatically active (Fig. 12A, 2 nd bar) whereas the control plate that was treated by esterase conjugated with the GpCpPuromycin did not bind the enzyme and had considerably lower activity (Fig. 12A, 1 st bar).
  • the residual esterase activity in this control experiment was probably due to unspecific adsorption of the esterase to the polystyrene surface.
  • Example 13 Immobilization of esterase 2 having incorporated a biotinylated puromycine-derivative on streptavidin coated glass surfaces
  • Example 14 Preparation of the plasmid pEst2_amb155 comprising a cDNA encoding an AGC 155 ⁇ TAG 155 -mutated esterase 2 (EST2) from Alicyclobacillus acidocaldarius
  • the mutated codons are bolded and the serine codon at position 155 of amino acid sequence of the esterase was changed to RF1 stop codon (TAG) mutation.
  • the PCR fragments were fused by another PCR using T7 promotor and T7 terminator primers.
  • the fused PCR product was digested with NcollSacl and ligated into Ncol/Sacl digested plVEX 2.3d vector.
  • the ligation mixture was transformed into E.coli strain XL-1 Blue.
  • the plasmid DNA was isolated from clones and sequenced before use.
  • the resulting plasmid pEst2_amb155 was used for in vitro translation.
  • a map of pEst2_amb155 is shown in Fig. 14, the corresponding nucleotide sequence is shown in Fig. 15 (SEQ ID NO: 41).
  • the plasmid was purified as described herein above (Ex. 7)
  • the used suppressor tRNA SerCUA was prepared as follows.
  • tRNA SerCUA The gene of tRNA SerCUA was constructed by PCR using primers tSer-amber1 (5'- GGAATTCTAATACGACTCACTATAGGAGAGATGCC-3'; SEQ ID NO: 48), tSer- amber2 (5'-GTCCGTTCAGCCGCTCCGGCATCTCTCCTATAGTG-3'; SEQ ID NO: 49), tSer-amber3 (5'-CTCCGGTTTTAGAGACCGGTCCGTTCAGCCGCTCC-3'; SEQ ID NO: 50), tSer-amber4 (5'-CCGGTA GAGTTGCCCCTACTCCGGTTTTA GA GA CC- 3'; SEQ ID NO: 51), tSer-amber5 (5'--
  • tSer- amber ⁇ (5'-AAGCTTGGATGGATCACCTGGCGGAGAGAGGGGGATTTGAAC-3'; SEQ ID NO: 53).
  • Bolded letters are T7 promotor and italic letters are the gene of tRNA SerCUA .
  • the mutated anticodon is underlined.
  • the conditions of the performed PCR were 95°C denaturation for 30 seconds, 50°C annealing for 30 seconds and 72°C polymerization for 30 seconds; 25 cycles were performed.
  • the primer concentration was about 1 nM.
  • DNTP concentration was 0.4 mM.
  • the sequence of suppressor tRNA is based on a tRNA Ser from E.coli (tRNA databank number DS 1660) with a CUA mutation from position 34 to 36.
  • the PCR product was cloned in a pGEM-T vector.
  • the resulting plasmid ptSer-amber was sequenced and used as a template for the following PCR.
  • the PCR was performed with primers tSer-amber1 and M13_rev (5'-CAGGAAACAGCTATGACC-3'; SEQ ID NO: 54).
  • the PCR product was digested with BstNI for a CCA end and used as the template for in vitro transcription.
  • Example 16 Depletion of the release factor 1 from E. coli in an E. coli cell-free translation system by precipitating and/or crosslinking said release factor 1 from E. coli with polyclonal antibodies against the release factor 1 of Thermus thermophilus and thereby increasing the incorporation of an (unnatural) amino acid delivered by aminoacyl suppressor tRNA CUA at an internal nonsense codon of an AGC 155 ⁇ TAG 155 -mutated esterase 2 (EST2) from Alicyclobacillus acidocaldarius
  • kits used for in vitro translation experiments within the present application were evaluation size transcription/translation kits from RiNA GmbH (Berlin, Germany) and the reaction was performed according to the manual provided by the supplier.
  • [ 14 C]L- Leucine (54 mCi/mmol) was added up to 160 ⁇ M along with leucine resulting in 0.5 mM total concentration.
  • the templates pEst2 and pEst2_amb155 were added up to 5 nM concentrations.
  • the reaction was performed at 37 °C with agitation. Aliquots, 3 ⁇ L, were withdrawn at different time intervals and the newly synthesized protein was determined by radioactivity measurement in 10% trichloroacetic acid precipitate. Protein composition was analysed by SDS-PAG].
  • the gels were fixed with 15% formaldehyde in 60% methanol and stained with Coomassie Blue G-250.
  • the dried gels were exposed to an imaging plate for radioactivity analysis with the Phosphorlmager SI (Molecular Dynamics, Sunnyvale, USA
  • in vitro translations were performed according to the protocols shown in example 11 with minor modifications. As an example a detailed protocol of the in vitro translation performed in the presence of anti-RF1 (T. thermophilus) antibodies and suppressor tRNA is listed below.
  • anti-RF1 T. thermophilus
  • reaction mixture should be incubated at 37°C for 2 hours with agitation (500 rpm). Aliquots can be withdrawn at any desired time intervals.
  • the standard in vitro translation system (without RF depleating agents (e. g. Antibodies against RF1 from Thermus thermophilus) or nonsense codon suppressing agents (e. g. puromycin derivatives and/or suppressor tRNAs) of the protocol listed above comprises the following ingredients:
  • each amino acid can be applied as a 14C amino acid with a concentration of 100 ⁇ M (e.g. 14C-leucine))
  • - Protease inhibitors aprotinin 10 ⁇ g/ml reaction mixture, leupeptin 5 ⁇ g/ml reaction mixture, pepstatin 5 ⁇ g/ml reaction mixture; and - Folic acid 50 ⁇ g/ml reaction mixture.
  • Example 14 Using the construct of Example 14 as a template for in vitro protein synthesis, the suppression of the amber codon was studied by SDS-PAGE of the full-length esterase production (Fig. 17B) and by measurement of the catalytic activity of the in vitro synthesized esterase (Fig. 17C).
  • Fig. 17B Translation of est2 mRNA(Ser-155) and est2 mRNA(amber-155) as measured by [ 14 C]leucine incorporation into polypeptide chain provides a protein of 34.4 and 17.3 kDa (Fig. 17B), respectively, approximately with the same efficiency (Fig. 17A).
  • the amber mutation in position 155 leads to complete termination and synthesis of 17.3 kDa protein void of esterase activity (Fig. 17B; lane 1 and Fig. 17C).
  • FIG 18 also demonstrates the effect of polyclonal antibodies raised against RF1 in promoting a suppression of an AUG amber triplet during E. coli in vitro translation.
  • FIG 16A the two DNA constructs, one with Ser 155 and the other with amber 155 are shown.
  • SDS-PAGE and the autoradiography of the 14 C-labelled products of in vitro synthesis are shown in Fig 18A.
  • the esterase acticity of the in vitro synthetised products is demonstrated in Fig 18B.
  • Esterase 2 from Alicyclobacillus acidocaldarius has an essential serine residue in position 155 of its sequence, that is part of the catalytic triad (Ser-His-Asp).
  • the hydroxyl of this serine side chain is the acceptor of the acyl residue during the hydrolysis of the ester bond.
  • the lane 4 of the SDS -PAGE experiment shown in Fig.18A demonstrates the synthesis of the active full-length protein when a DNA with an normal AGC coding for Ser 155 is present.
  • the replacement of this triplet by an amber stop codon UAG leads to a production of a inactive polypeptide with 154 amino acids (Fig 18A, lane 1 , Fig 18B, column 1). This is a result of the recognition of UAG triplet by endogenous RF1 and hydrolysis of peptidyl-tRNA.
  • the concentration of RF1 becomes about 50 fold lower as compared with the situation in vivo. It follows, that the average final concentration of a single aminoacyl-tRNA isoacceptor and RF1 in the in vitro translation mixture is about 1 ⁇ M and 20 nM, respectively.
  • the need for high Ser- tRNA Ser(CUA) concentrations to compete for RF1 probably reflects the different affinity for the ribosomal A-site of these alternative substrates.
  • est2 mRNA(amber 155) as a template and the possibility to deactivate the endogenous RF1 in the in vitro translation system by RF1 antibodies permits an optimal adjustment of RF1 and tRNA Ser(CUA) concentrations to achieve a complete suppression and at the same time a maximal retention of enzymatic activity of the esterase.
  • Example 17 Preparation of protein arrays by using the ,,on spot" in vitro protein biosynthesis technology of the present invention
  • the DNA that contains
  • a termination triplet is covalently attached to a solid surface (see, Fig 20).
  • a solid surface Preferentially, transparent glass microscope slides coated by streptavidine are used and the DNA is attached via an 5'-biotin tag.
  • Other method of DNA immobilization by photochemical or chemical methods are also feasible.
  • the surface on which the DNA is attached is overlaid by a solution containing the complete in vitro translation system.
  • a C-terminal puromycin-dependent tagging system of the polypeptide is included in the bacterial in vitro translation extract.
  • Biotin coupling the puromycin analogue is the obvious choice to achieve such immobilisation on streptavidin-coated solid plates. But alternative covalent coupling is also highly feasible. It was experimentally proven that puromycin analogue is also active in immobilized form provided the length of the linker to the solid surface is appropriate. It is, therefore, not important if the puromycin analogue carrying a biotin tag becomes immobilized prior to is attachment to the C-terminus of the protein. The "on spot" in vitro protein biosynthesis technology relies on selective depletion of a particular release factor from the translation extract, which is achieved by polyclonal antibodies against RF1 and RF2 in the case of UAG and UGA termination codons, respectively.
  • polyclonal antibodies against orthologous release factors from Thermus thermophilus are used.
  • the biotin is covalently attached to a puromycin analogue PupC 5 .
  • PupC 5 a puromycin analogue
  • Important feature of the system is that as a result of RF-depletion the biotin-puromycin, or chemically active puromycin analogue, are incorporated only into the full-length polypeptide.
  • the transcription- translation coupling and the puromycin-analogue tagging of the protein will assure the "on spot" topospecific synthesis of the translation product on the two-dimensional solid support (see, Fig 20).
  • each immobilised DNA matrix will be exponentially multiplied by transcription and translation in close vicinity of the location on which the particular DNA sequence was attached.
  • a protein array can be also constructed by microcompartmentisation of the DNA matrix on the solid support.
  • the gene of the expressed protein can be coupled with a reporter gene linked via a cleavable protease site.
  • a reporter gene linked via a cleavable protease site.
  • the esterase 2 from Alicyclobacillus acidocaldarius, a single chain, thermostable enzyme is used as an sensitive reporter group, but other types of labelling using radioactive or spectroscopic reporter groups are possible.
  • the described method is suitable for:
  • a similar system as described above can be used for in vitro selection of proteins from a library of mutated DNA matrices.
  • the PCR product that contains the modules depicted in Fig. 20. and carries a biotin tag, or a reactive group on the 5'-end.
  • the coding part of the immobilised matrix DNA contains a random sequence.
  • the correct sequence coding for the required protein is then selected. Many of the sequences combinations will cause terminations and synthesize short polypeptide. These sequences will be eliminated on the bases of missing esterase activity.
  • the esterase reporter gene is located on the C-terminal site of the analysed protein, therefore each termination event in the analysed gene will result in inactive esterase.
  • Enzymatic activity of the esterase can be detected with sensitivity better then 10 3 molecules/spot. DNA in the positive spots will be multiplied by PCR and 5'— biotinylated. After appropriate dilution the "Winner” DNA is placed on the new plate and the procedure is repeated. This can be repeated several times. In the next cycles a selection principle directed to a biochemical property of the N-terminaly placed protein ("active esterase" in Fig 20) is applied. Finally, the "winner” DNA sequences are cloned and sequenced.
  • the described SPOT-display method relies on following features:
  • Proteins are coded by a few DNA molecules located on particular spot. This is achieved by maximal possible dilution of the DNA used for attachment to the solid support,
  • Puromycin analogue is immobilized via an appropriate long linker to the same location on the solid support as is the DNA matrix,
  • Immobilized Puromycin analogue is specifically incorporated in the full-length polypeptide in the position determined by stop codons, under condition of depletion of the release factors from the in vitro translation system.
  • the describe SPOT-display is suitable for protein engineering:
  • the RF1 responsible for termination at the UAA and the UAG stop codons was inactivated in the in vitro translation system from E. coli by rabbit antibodies raised against RF1 from Thermus thermophilus as described herein.
  • plasmid DNA that encoded mRNA for synthesis of the esterase from Alicyclobacillus acidocaldarius (Manco 1998 loc. cit.) as described herein and terminated by UAG stop codon was used.
  • the functionally active enzyme was produced up to a concentration of 200 ⁇ g/ml. Its C-terminal labeling was monitored by incorporation of [32P]pGpCpPuromycin (Fig.
  • the conjugation yield was determined as a ratio between the amount of active enzyme produced in the system that was calculated from esterase specific activity and the amount of puromycin derivative attached to the full length product calculated on the base of 32 P radioactivity by the Phosphoimager software.
  • the antibody concentration range in which the puromycin-mediated termination is turned on is very narrow (Fig. 21A and B) and correlates well with the concentration of RF1 at which an efficient incorporation is blocked via addition of purified E. coli RF1 (Fig. 21 C and D).
  • Figure 21 shows that only at high anti RF1 antibody concentration [32P]pGpCpPuromycin starts to be incorporated efficiently into full-length esterase.
  • Taq polymerase was from Qiagen (Hilden, Germany), T4-DNA-I_igase from Promega (Mannheim, Germany), Factor Xa protease and restriction enzymes were from NewEngland Biolabs (Frankfurt, Germany).
  • Fast Blue BB Salt, p-Nitrophenyl acetate and ⁇ -Naphthyl-acetate were from Fluka (Steinheim, Germany).
  • 5-(and 6-) Carboxy- 2',7'-dichlorofluoresceine diacetate was from Molecular probes (Eugene, USA).
  • Other analytical grade chemicals were obtained from Roth (Karlsruhe, Germany).
  • Radioactive [ 14 C]leucine 54 mCi/mmol was from Amersham, Life Sciences (Freiburg, Germany).
  • the present invention refers to the following nucleotide and amino acid sequences:
  • Nucleotide sequence encoding releasefactor 1 from Thermus thermophilus atgctggacaagcttgaccgcctagaggaagagtaccgggagctggaggc gctcctccgacccggaggtgctgaaggacaaggggcgctaccagagcc tctcccgccgctacgccgagatgggggaggtgatcggcctcatccgggag taccggaaggtgctggaggacctggagcaggcggaaagccttcttgacga cccgagctcaaggagatggccaaggcggagcgggaggccctctcgccc gcaaggagatggccaaggcggagcgggaggccctctcgccc gcaaggagatggccaaggcggag
  • ERF1_RABIT Eukaryotic peptide chain release factor subunit 1
  • Eukaryotic peptide chain release factor subunit 1 (Eukaryotic release factor 1) - Oryctolagus cuniculus (Rabbit).
  • SEQ ID No. 17 Nucleotide sequence encoding Eukaryotic polypeptide chain release factor3 (Fragment) - Oryctolagus cuniculus (Rabbit) ctggcggcggcggccgaggcccagcgtgaccacctcagcgcggccttcagccggcagctcaacgccaaacctttcgtgcccaac gtccacgcgccg cggagttcgtaccgtctttcctgcggggcccggcccgcctccagcccccggctggcgcgcggcaacaaccacggagcgggcagcgtcg cgtcg cgccggcaacaaccacggagcgggcagcgtcg cgcgcgccggcaacaaccacggagcgggcagcgtcg c
  • Eukaryotic peptide chain release factor subunit 1 (Eukaryotic release factor 1) - Drosophila melanogaster(Fruitfly).
  • eRF1 Eukaryotic peptide chain release factor subunit 1
  • Eukaryotic peptide chain release factor subunit 1 (Eukaryotic release factor 1) - Drosophila melanogaster (Fruit fly).
  • Eukaryotic peptide chain release factor subunit 1 (Eukaryotic release factor 1) (Omnipotent suppressor protein 1) - Saccharomyces cerevisiae (Baker's yeast).
  • Eukaryotic peptide chain release factor subunit 1 (Eukaryotic release factor 1) (Omnipotent suppressor protein 1) - Saccharomyces cerevisiae (Baker's yeast).
  • Eukaryotic peptide chain release factor GTP-binding subunit (ERF2) (Translation release factor 3) (ERF3) (ERF-3) (Omnipotent suppressor protein 2) (G1 to S phase transition protein 1) - Saccharomyces cerevisiae (Baker's yeast).
  • Eukaryotic peptide chain release factor GTP-binding subunit (ERF2) (Translation release factor 3) (ERF3) (ERF-3) (Omnipotent suppressor protein 2) (G1 to S phase transition protein 1) - Saccharomyces cerevisiae (Baker's yeast).
  • Saccharomyces cerevisiae (Baker's yeast).
  • SEQ ID No. 31 Nucleotide sequence encoding Translation termination factor SUP35 (Fragment) - Saccharomyces cerevisiae (Baker's yeast).
  • amino acid residues indicated with X are methionine residues (Met (M)) as encoded by the corresponding codon triplet "ATG” as shown in SEQ ID NO:1.
  • the amino acid sequence corresponding to SEQ ID NO: 39 and having methionine residues (Met (M)) at amino acid position 53, 64, 210 and 211 is shown in SEQ ID NO: 55 of the sequence listing.

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Abstract

The present invention relates to a ceil-free translation system comprising a nonsense-codon suppressing agent and an anti-release factor antibody which precipitates an/ or crosslinks a release factor in said cell-free translation system. The present invention further relates to a kit comprising said cell free translation system. Moreover, the present invention relates to an anti-release factor antibody directed against release factor of Thermus thermophiles. Furthermore, the present invention relates to the use of an anti-release factor antibody for the preparation of a celi-free translation system. Moreover, the present invention relates to a method for the production of an anti-release factor antibody. Furthermore, the present invention provides for a method for the production of alloproteins. Further, the present invention relates to a puromycin derivative having a residue covalently attached directly or via a linker to one of its elements selected from the group consisting of the element N4 of the cytosine-residue of an 51 attached cytidine-residue and the element C5 of the cytosine-residue of an 5' attached cytidine-residue. Moreover, the present invention relates to the use of said puromycin derivative in the cell-free translation system of the present invention, in the method for the production of alloproteins of the present invention. The invention also provides for specific kits described herein.

Description

Cell-free translation system for the production of (chemically) modified proteins
The present invention relates to a cell-free translation system comprising a nonsense-codon suppressing agent and an anti-release factor antibody which precipitates and/or crosslinks a release factor in said cell-free translation system. The present invention further relates to a kit comprising said cell free translation system. Moreover, the present invention relates to an anti-release factor antibody directed against release factor 1 of Thermus thermophiles and, inter alia, obtainable by eliciting an in vivo humoral response against release factor 1 from Thermus thermophilus or a fragment thereof in a non-human vertebrates. Furthermore, the present invention relates to the use of an anti-release factor antibody for the preparation of a cell-free translation system. Moreover, the present invention relates to a method for the production of an anti-release factor antibody comprising the steps of eliciting an in vivo humoral response against release factor 1 from Thermus thermophilus or a fragment thereof in a non-human vertebrate. Furthermore, the present invention provides for a method for the production of alloproteins, comprising the step of translating RNA into a translation product in the cell-free translation system of the present invention. Further, the present invention relates to a puromycin derivative having a residue covalently attached directly or via a linker to one of its elements selected from the group consisting of the element N4 of the cytosine- residue of an 5' attached cytidine-residue and the element C5 of the cytosine-residue of an 5' attached cytidine-residue. Moreover, the present invention relates to the use of a puromycin derivative having a residue covalently attached directly or via a linker to one of its elements selected from the group consisting of the element N4 of the cytosine-residue of an 5' attached cytidine-residue and the element C5 of the cytosine-residue of an 5' attached cytidine-residue, in the cell-free translation system of the present invention, in the method for the production of alloproteins of the present invention. The invention also provides for specific kits described herein. Protein conjugates, e.g. glycoproteins, nucleoproteins, phosphoproteins and lipoproteins, are involved in numerous cellular functions. They play an important role in macromolecular recognition, signal transduction, regulation of enzymatic activities and gene expression and in cellular localization of proteins. Therefore, there is a demand for protein conjugates for numerous applications in modern bioscience (Eisele (1999), Bioorg. Med. Chem., 7, 193-224) (Niemeyer (2002), Trends Biotechnol., 20, 395-401). Moreover, in biochemical praxis there is a need for production of alloproteins, i.e. proteins that, in addition to 20 natural proteinogenic amino acids, contain amino acid with side chains that naturally do not occur.
For instance, the attachment of a nucleic acid with a specific sequence to proteins allows to link properties of these two distinct groups of biopolymers in one molecule. The resulting conjugates can be used for variety of applications including preparation of synthetic enzymes (Corey (1987), Science, 238, 1401-1403), gene therapy (Zanta (1999), Proc. Natl. Acad. Sci. U.S.A., 96, 91-96), construction of protein microarray (Niemeyer (1994), Nucleic Acid Res., 22, 5530-5539), creation of molecular scale devices (Keren (2002), Science, 297, 72-75), and development of immunological assays (Niemeyer (2003), Nucleic Acids Res., 31 , e90). In the light of the importance of protein-nucleic acid conjugates, several different approaches for their production were reported. The direct attachment of an oligonucleotide to an exposed cysteine residue of a protein (Howorka (2001), Nat. Biotechnol., 19, 636-639) is restricted by the properties and function of the accessible cystein residues. Alternative approaches utilize bifunctional cross-linking reagents to perform direct covalent attachment of an oligonucleotide to a protein (Schweitzer (2002), Nat. Biotechnol., 20, 359-365). These strategies, based on random chemical modification, usually lead to heterogeneity of the final product which is a considerable disadvantage when using said products in biochemical and biomedical praxis. A specific conjugation of proteins with variety of functional groups can be achieved by ligation of chemically synthesized modified peptides with target polypeptides leading to recombinant proteins (Cotton (1999), Chem. Biol., 6, R247-R256) (Goody (2002), Chembiochem., 3, 399-403) (Eisele (1999), Bioorg. Med. Chem., 7, 193-224). Thus, recombinant proteins containing C-terminal α-thio-ester can be conjugated with cysteinyl- deoxyribonucleic acids by protein ligation leading to oligonucleotides specifically attached to C-terminus of recombinant protein (Takeda (2004), Bioorg. Med. Chem. Lett., 14, 2407-2410).
Despite of the importance of protein conjugates (e.g., protein-nucleic acid conjugates), a convenient and universal covalent conjugation procedure was not yet developed. This problem was addressed for several times within the art. One approach aimed the suppression of nonsense-codons by a suppressor tRNA, which is aminoacylated by an unnatural amino acid during in vitro translation, and thereby covalently attaching different amino acids to the synthesized proteins. The suppression of the nonsense-codons was either achieved by the expansion of the genetic code (Wang (2002), Chem. Commun. (Camb). (1):1-11) or by the use of the low molecular weight antibiotic puromycin.
In the first case, one of the three available triplets coding for termination of translation (UAG, UAA and UGA) are "recorded" for recognition by suppressor tRNAs that are able to read one of these stop codons. Usually the UAG codon that is recognized by the release factor 1 (RF 1) is selected for recognition by a suppressor tRNA (tRNASuP) chemically aminoacylated, i.e. not enzymatically, by an unnatural amino acid. Said strategy was embarked for the production of different alloproteins. In the latter case, puromycin was incorporated into the C-terminus of the protein and is used as a vehicle to transfer a variety of functional groups into the (in vitro) synthesized protein.
However, both approaches mentioned above where limited in the fact that the corresponding synthesis efficiency appear to be low and not satisfactory.
As a rule, when stop codons appear in the ribosomal A-site there is no aminoacyl- tRNAΕF-Tu.GTP ternary complex available to read the termination triplet. Instead a protein, in particular a class I release factor (RF), decodes the termination triplet and initiates the peptide hydrolysis from the P-site bound peptidyl-tRNA and the peptide release (Kisselev (2003), EMBO J 22, 175-182). This implies that the affinity of class I RFs to the A-site of termination codon-programmed ribosomes must be considerably higher then the affinity of near-cognate aminoacyl-tRNAs to this site (Freistroffer (2000), Proc. Natl. Acad. Sci. U. S. A 97, 2046-2051). It follows that the concentrations of RF and aminoacyl-tRNAs in the cell has to be precisely balanced in order to achieve a precisely controlled termination (Adamski (1994), J. MoI. Biol 238, 302-308). At concentrations of RF below the normal cellular level, the chance for aminoacyl-tRNA with a near-cognate anticodon to be bound to "hungry" termination codon-programmed ribosomes and to suppress the termination signal will increase. On the other hand, in the presence of RF1 at normal cellular concentration, if the concentration of individual aminoacyl-tRNA-EF-Tu.GTP increases above a tolerated limit, the chance for misreading at near-cognate codons during in vitro translation, including suppression of stop codons, will rise (LaRiviere (2001), Science 294, 165- 168). As noted earlier, the concentration of RF1 does not regulate exclusively the termination but at the same influences the fidelity of translation (Jorgensen (1993), J. MoI. Biol. 230, 41-50).
There are also other factors that influence the probability of suppression as e.g. codon context (Pavlov (1998), J MoI. Biol 284, 579-590), mutations in ribosomal RNAs (Arkov (2002), J Bacteriol. 184, 5052-5057) tRNA structure (Smith (1989), J MoI Biol 206, 489-501) and modification (Baumann (1985), Eur. J. Biochem. 152, 645-649), structure of the nascent peptide and its interaction with the peptide exit site (Tenson (2002), Ce// 108, 591-594).
Mechanism of termination of polypeptide biosynthesis at UAG, UAA and UGA stop codons and suppression of these codons by suppressor tRNAs is an intensively investigated topic related to structure of translation apparatus (Klaholz (2003), Nature 421, 90-94), regulation of gene expression on the translational level, natural incorporation of seldom amino acids into polypeptides (Heider (1992), EMBO J. 11, 3759-3766.; Hao (2002), Science 296, 1462-1466) and practical importance for the broad application of cell-free protein synthesis for preparation of native, modified and conjugated polypeptides (Miyamoto-Sato (2000), Nucleic Acids Res. 28, 1176-1182.; Noren (1989), Science 244, 182-188).
Biosynthesis of proteins in all living organisms is based on peptidyl transferase reaction catalyzed by the ribosome. A nucleophilic attack on the carbonyl group of the ester bond of the peptidyl-tRNA by the amino group of the aminoacyl-tRNA results in formation of an amide (peptide) bond (Fig. 1A). At concentrations greater than 10 μM the antibiotic puromycin, that by its structure resembles the aminoacylated 3'-terminal adenosine of the aminoacyl-tRNA (Fig. 1 B), can compete with aminoacyl-tRNA.EF-Tu.GTP ternary complex for the ribosomal A-site. In such case it serves as a low molecular weight acceptor substrate in peptidyl transferase reaction leading to formation of puromycin-peptide that is released from ribosomes (Starck (2002), RNA., 8, 890-903). At low concentration (0,04 μM) puromycin derivatives possess negligible inhibitory effect on elongation, but instead, they are incorporated into C-terminus of the polypeptide (with low efficiency) when the stop codon is presented in the A-site (Miyamoto-Sato (2000), Nucleic Acids Res., 28, 1176-1182). The efficiency of this puromycin-mediated termination is, however, very low.
Yanagawa and coworkers described said (inefficient) incorporation of puromycin, at positions mediated by truncated mRNAs (Nemoto (1999), FEBS Lett, 462, 43-46) or at positions coded by stop codons (Miyamoto-Sato (2000), Nucleic Acids Res., 28, 1176-1182) into proteins and used this approach for C-terminal labeling of proteins with biotin and fluorescence reporter groups. They found out that the C-terminal incorporation of puromycin derivatives is stimulated in the absence of a stop codon in the mRNA, particularly for proteins longer than 14 kD (Nemoto (1999), FEBS Lett., 462, 43-46) and that it is dependent on the concentration of RFs (Miyamoto-Sato (2000), Nucleic Acids Res., 28, 1176-1182). This indicates that the RFs, programmed for binding to the ribosomal A-site by stop codons, compete with puromycin for the A- site binding (Fig. 1).
Even though, the in vitro technology for radioactive (Miyamoto-Sato (2000), Nucleic Acids Res., 28, 1176-1182) or fluorescence (Nemoto (1999), FEBS Lett., 462, 43-46) labelling of the C-terminus of proteins with puromycin analogues has been reported by Yanagawa and co-workers (Doi (2002), Genome Res., 12, 487-492) (Kawahashi (2003), Proteomics., 3, 1236-1243), the efficiency of said labelling was extremely low.
It was previously suggested that inactivation of RFs by RNA aptamers (Szkaradkiewicz (2002), FEBS Lett. 514, 90-95.; Games, J. (2000) RNA. 6, 1468- 1479) or temperature deactivation of thermosensitive RF1 (Short (1999) Biochemistry 38, 8808-8819) increases the efficiency of suppression of nonsense-codons by suppressor tRNA. However, similar as in the case of puromycin incorporation, the efficiency of said suppression by suppressor tRNA is also low, due to the competition with normal termination at UAG codons by remaining endogenous RFs.
Furthermore, naturally occurring suppression events, as the incorporation of selenocysteine (Heider (1992), EMBO J. 11, 3759-3766) or pyrolysine (Hao (2002) Science 296, 1462-1466) into polypeptides at UAG triplets, are complicated and directed by special suppressor tRNAs that carry the respective amino acid and specific structural elements of mRNA near the recoding UAG site. In the case of selenocysteine incorporation additional accessory protein, an analogue of elongation factor Tu (Forchhammer (1990), J Biol Chem 265, 9346-9350), is involved in the process.
The failure of the attempts of the prior art to develop a highly efficient, convenient and universal system for the production of protein conjugates, is a consequence of the fact that one important restriction of the technology was never solved: A precise balance between nonsense-codon suppressing agents (e.g. suppressor tRNA, puromycin-derivatives), RFs and aa-tRNA EF-Tu.GTP ternary complex has to be obeyed to satisfactorily apply this technology. On the other hand, the concentration of puromycin (-analogues) cannot be increased beyond a critical level because of elongation inhibition. The concentration of suppressor tRNA cannot be increased beyond a critical level because of erroneous, premature termination. Accordingly, a loss of activity of the (in vitro) synthesized proteins would occur or the synthesis of said proteins would not be possible at all. On the other hand, both nonsense-codon suppressing agents cannot be decreased among a critical level, since the competition with RFs and therefore, the incorporation of puromycin (-analogues) and/or unnatural amino acids would become inefficient.
Taken together, the efficiency of nonsense-codon suppression in known (in vitro) translation-systems is (extremely) weak and therefore, the economic production of protein-conjugates and alloproteins by utilizing these systems is almost impossible. Thus, the technical problem underlying the present invention is the provision of means and methods for the amelioration of translation efficiencies in particular the translation efficiencies for the production of (chemically) modified proteins and/or alloproteins.
The solution to the above technical problem is achieved by providing the embodiments characterized in the claims.
Accordingly, the present invention relates to a cell-free translation system comprising a nonsense-codon suppressing agent and an anti-release factor antibody which precipitates and/or crosslinks a release factor in said cell-free translation system.
The present invention solves the above identified technical problem since, as documented herein below and in the appended examples, the cell-free translation system of the present invention provides for at least 50% suppression, more preferably at least 60% suppression, more preferably at least 70% suppression, more preferably at least 80% suppression, even more preferably at least 90% suppression and most preferably at least 98% suppression. As shown in the appended examples, the present invention even provides for a cell-free translation system, wherein the nonsense-codons are completely suppressed, i.e. up to nearly 100% suppression can be achieved.
Suppression of nonsense-codons can be measured as shown in the appended examples, in particular by detecting the incorporation of nonsense-codon suppressing agents in proteinaceous material obtained in said in-vitro translation systems. (An example may be the incorporation of radioactively labelled puromycin or derivatives thereof.)
As mentioned above, the inventive use of anti-release factor (RF) precipitating and/or crosslinking antibodies provides for nearly 100% suppression of nonsense-codons. In contrast, the use of aptamers directed against the same release-factor only provides for a portion of such a suppression, even if aptamers are present in excess. The reported and measured efficiency of translation systems of the prior art lays in values of about up to 30 % suppression of nonsense-codons. In context of the present invention it was surprisingly found that the use of anti-RF antibodies, i. e. antibodies capable of precipitation and/or crosslinking a RF leads to highly-efficient suppression of nonsense-codons (by nonsense-codon suppressing agents) and, thereby, to efficient in vitro synthesis values. In particular in the in vitro synthesis of modified, preferably chemically modified proteinaceous products, e. g. chemically modified products and/or alloproteins, highly efficient synthesis yields may be obtained with the cell free translation system of the present invention and the methods provided herein. Accordingly, the present invention provides, inter alia, for a system for coupled in vitro protein synthesis, capable for specific and highly-efficient production of proteins, in particular of proteins comprising chemical modifications and/or additions (C-terminal and/or internal). The system provided herein is particularly useful in the synthesis of (chemically) modified proteins and/or alloproteins.
In context of the present invention, the meaning of the term "protein(s)" also includes "peptide(s)" or "polypeptide(s)". The meaning of the terms "protein(s)", "peptide(s)" or "polypeptide(s)" are well known in the art (see, e.g., Stryer (1995), Biochemistry, 4th edition). As known in the art, the term "peptide" comprises joined amino acid residues, whereby the alpha-carboxyl group of one amino acid is joined to the alpha- group of another amino acid by a peptide bond (amide bond); see also Stryer ((1995), loc. cit.). In accordance with the invention, the term "peptide(s)" comprises any such joined amino acid residues, whereby at least three, preferably at least five, most preferably at least seven amino acids (amino acid residues) are linked via said peptide bond (amide bond). The term polypeptide comprises, in accordance with this invention, at least 15 joined amino acid residues, more preferably at least 20 amino acid residues. Accordingly, joined amino acid residues comprising 3 to 14 amino acid residues are to be considered in accordance with this invention as "peptide" whereas joined amino acid residues comprising 15 or more amino acid residues are considered as polypeptides. The term "protein" is used as synonym with the term "polypeptide", whereas the term "protein" also may comprise a specific biological, biochemical or pharmaceutical function exerted by said protein. However, the person skilled in the art is aware that a protein is a polypeptide. The terms "protein", "peptide" and "polypeptide" also comprise molecules comprising at least one unnaturally occurring amino acid residue or at least one unusual amino acid residue and is not limited to proteinaceous structures comprising the twenty normally occurring amino acid residues; see also Stryer ((1995), loc. cit.).
The solution of the afore-mentioned technical problem was achieved by almost exhaustive deactivation and/or inactivation of one kind of competitors for nonsense- codons, the RFs. Said inactivation of RFs in an in vitro translation system leads to a very favourable situation that the ribosomal A-site, programmed by a stop codon, becomes a specific/easy target for nonsense-codon suppressing agents (for example the antibiotic puromycin and/or suppressor tRNA). In the absence of active RF, nonsense-codon suppressing agents can be used as vehicles to carry chemical residues (for example (unnatural) amino acids, reporter groups, affinity tags or even large oligomeric structures) to be linked with a polypeptide.
As pointed out above, it was surprisingly found that an addition of RF specific antibodies to a cell-free translation system leads to dramatic increase of nonsense- codon suppression in the presence of nonsense-codon suppression agents. As a consequence, nonsense-codon suppressing agents can also be employed to efficiently carrying chemical residues to proteinaceous structures (proteins) synthesized. Said "chemical residues" can then be efficiently covalently linked to the protein structure. Accordingly, with the present invention, "unnatural" proteins and/or alloproteins may be successfully synthesized.
The present invention allows to use conventional coupled transcription/translation systems as a basis for high yield production of alloproteins. However, the conventional systems have, in accordance with this invention, to be further modified by a) the addition of an anti-RF antibody (for example a serum directed against the corresponding RF) and b) the addition of nonsense-codon suppressing agents.
The term "nonsense-codon suppressing agent" as used herein is known in the art and relates to an agent that is capable to bind to the A-site of a ribosome programmed by a stop codon. Said stop codons are known in the art and may be UAA, UAG or UGA, preferably, UAG. The nonsense-codon suppressing agent itself may be covalently bound to the elongating peptide-chain or may be delivering a substance that is bound to the elongating peptide chain. Said nonsense-codon suppressing agent may prevent normal termination accomplished by release factors or termination factors or said nonsense-codon suppressing agents may replace normal termination accomplished by release factors or termination factors. Preferably, said nonsense-codon suppressing agent that delivers a substance to be bound to the elongating peptide-chain prevents normal termination. Said nonsense- codon suppressing agent, being itself covalently bound to the elongating peptide- chain, replaces normal termination. The nonsense-codon suppressing agent, delivering the substance to be bound to the polypeptide-chain, may be a aminoacyl- tRNA, preferably a suppressor aminoacyl-tRNA, more preferably a suppressor aminoacyl-tRNA(CUA). The nonsense-codon suppressing agent to be covalently bound to the polypeptide chain may be, inter alia and preferably, puromycine or a derivative thereof as defined herein below.
The term "anti-RF antibody", in particular "anti-RF1 antibody" as employed herein refers to an antibody, a plurality of antibodies and/or a serum comprising such antibodies which is/are able to specifically bind to, interact with and/or detect RFs, preferably RF1 , more preferably RF1 from E. coli (e.g. as shown in SEQ ID NO: 6) or a fragment thereof. In context of the present invention, said "anti-RF antibody" must be capable of precipitating (in the in vitro system) the RF and/or must be capable of crosslinking said RF. The "precipitation" and/or crosslinking" leads to an inactivation of the RF, inter alia, due to the formation of larger RF- antibody complexes. The term "precipitates and/or crosslinks", accordingly, refers to the capability of an anti-release factor antibody to bind and to inactivate a release factor. Therefore, said binding leads to an inactivation of said release factors which is equivalent of a depletion of said release factor (from cell-free translation systems). The term "inactivation" refers to making said release factors incapable to bind to the A-site of the ribosome and thereby incapable to cause termination of the peptide-chain and its release from the ribosomal complex. The precipitating and/or deactivating activity of anti-RF antibodies, in particular but not limiting polyclonal antibodies or sera containing those, can, inter alia, be measured by the residual RF activity in the in vitro translation system after adding of said anti-RF antibodies, by testing the hydrolysis of a peptide from peptidyl-tRNA located in the P-site (Freistroffer (2000), Proc Natl Acad Sci U S A. 97, 2046-51) or by a gel electrophoresis followed by Western blotting, which is a common laboratory praxis. Thereby, e. g. the formation of complexes between the RFs and anti-RF antibodies can be measured by measuring complex formation, for example in native gels. Here, individual proteins/polypeptides/peptides and complexes may be detected in form of either single individual bands or in form of complexes-formed broader bands in high molecular weight regions. Said banding pattern may be compared in gels comprising reducing agents (like DTT) and in gels without such agents. The corresponding technology is standard and known in the art; see, e.g. Sambroock (2001. Molecular Cloning, Ed 3.). Moreover, the precipitating and/or deactivating activity of anti-RF antibodies (like, inter alia, polyclonal antibodies or sera containing them) can be measured by complementing the depletion of RF through said antibodies or sera by addition of certain amounts of RF protein. Such a measurement method is exemplified in the appended examples (Example 20, Figure 21).
Corresponding antibodies may easily be prepared as demonstrated in the appended examples and as known in the art. Said antibodies and/or sera may, inter alia, be prepared by immunization of a non-human vertebrate with purified and/or recombinants produced "release factors". In the appended examples, it is documented how, for example a polyclonal serum against release factor 1 (RF1) of Thermus thermophilus (T. th; SEQ ID NO: 2) can be routinely and reliably prepared. In the corresponding example, a heterologously expressed, recombinantly produced RF1 was used in the immunization protocol. The preparation of antibodies, either monoclonal or polyclonal, is well known in the art; see, inter alia, Harlow/Lane ("Antibodies: A laboratory manual" (1988), CSHL, New York). The person skilled in the art readily in the position to deduce whether an antibody and/or antibody molecule or a serum directed against a given release factor is capable of precipitating and/or crosslinking said release factor.
The term "anti-RF antibody" also relates to a serum, in particular a purified serum, i.e. a purified polyclonal serum. The antibody molecule is preferably a full immunoglobulin, like an IgG, IgA, IgM, IgD, IgE, IgY (for example in yolk derived antibodies). The term "antibody" as used in this context of this invention also relates to a mixture of individual immunoglobulins. Furthermore, it is envisaged that the antibody/antibody molecule is a fragment of an antibody, like an F(ab), F(abc), Fv Fab' or F(ab)2. Furthermore, the term "antibody" as employed in the invention also relates to derivatives of the antibodies which display the same specificity as the described antibodies. Such derivatives may, inter alia, comprise chimeric antibodies or single-chain constructs. Yet, most preferably, and as shown in the examples, said "anti-RF antibody" relates to a serum. Also a purified (polyclonal) serum and, preferably, to a non-purified crude polyclonal serum. The antibody/serum is obtainable, and preferably obtained, by the method described herein and illustrated in the appended examples or by other methods known in the art.
As exemplified in the experimental part, said anti-RF antibody, in particular said anti- RF1 antibody, may specifically deplete one particular RF (e.g. RF1 (e.g. having the amino acid sequence of SEQ ID NO: 6)) keeping (an-)other RF(s) (e.g. RF2 (e.g. having the amino acid sequence of SEQ ID NO: 8)) active. In this case, a nonsense- codon suppressing agent (e.g. suppressor tRNA) can bind to the corresponding STOP-codon (e.g. UAG) of the first RF (e.g. RF1) and the second RF (e.g. RF2) is still capable to accomplish normal termination at the corresponding second STOP- codon (e.g. UGA). Said first STOP-codon may be an artificial STOP-codon lying inside of the open reading frame of a mRNA to be translated. Said second STOP- codon may lie at the end of said open reading frame.
The term "release factor" as used herein relates to any factor(s) that is/are capable to bind to the A-site of a ribosome programmed by a stop codon, whereby the stop codon is defined as mentioned herein above. By binding to said A-site, said release factor causes termination of the elongation of a peptide-chain during translation process, and thereby leads to a release of the nascent peptide-chain from the ribosomal complex. Preferably, the term "release factor" refers to release factors that are contained in cell-free translation systems. In context of the present invention, the term "release factor" also relates to a fragment of a release factor as defined herein. The term "fragment" (of a release factor) as used herein relates to fragments of a length of at least 30, at least 40, at least 50, more preferably at least 60, ever more preferably at least 65 amino acid residues of a (native) RF as defined herein. The amino acid sequence of RFs are known in the art and also specified herein below. Preferably, said fragment comprises at least such stretch of amino acids that (polyclonal) antibodies may be raised against this fragments and that these obtained antibodies are capable to precipitate and/or crosslink a release factor in a cell-free translation system.
The term "cell-free translation system" refers to cell-free translation systems commonly employed in in vitro synthesis approaches for proteins. In general, cell- free translation system are known in the art (e.g. Spirin (1990), American Society for Microbiology, 56-70; Stiege (1995), J. Biotechnol. 41:81-90; Zubay (1973), Imm. Rev. Genet. Vol. 7, page 267; Pelham (1976), Eur. J. Biochem. Vol. 131 , page 289; WO 9307287; EP 1254962; US 5571690; EP 1251168) and also further defined herein below. Yet, the cell-free translation systems known do not comprise an anti-release factor antibody and do not comprise a nonsense-codon suppressing agent. Accordingly, known cell-free translation systems do not comprise the two essential parts of the cell-free translation system of the present invention a) an anti-RF antibody capable of inactivating the release factor and b) a nonsense-codon suppressing agent.
In cell-free translation systems, the presence of active release factors competes with nonsense codon suppressing agents for the binding to the nonsense-codon programmed A-site. As the affinity of active release factors to the nonsense-codon programmed A-site is very high, the binding of nonsense codon suppressing agents is impaired/inhibited, even when small amounts of active release factors are present in cell-free translation systems.
Consequently, the basic requirement for an effective binding of nonsense codon suppressing agents to the ribosomal A-site, and, accordingly, for the attachment of an substituent, potentially an additional substituent, to the nascent peptide chain, is an exhaustive depletion of active release factors in the systems. In cell-free translation systems of the prior art, there is always a considerable amount of active release factors, and therefore an attachment of an (additional) substituent to the nascent peptide chain is ineffective.
In contrast, in cell-free translation systems as described herein, a nearly complete depletion of active release factors is achieved. Said complete depletion is obtained by adding an anti-release factor antibody to the cell-free translation system. Accordingly, one advantage of the invention is that an effective attachment of an (additional) substituent to the nascent peptide chain can be obtained.
Cell-free translation systems are made from cell-free extracts produced from prokaryotic or eukaryotic cells that contain all the necessary components to translate RNA (in particular mRNA) into protein. Cell-free extracts can be prepared from prokaryotic cells such as E. coli cells (e.g. Zubay (1973), Imm. Rev. Genet. Vol. 7, page 267) and from eukaryotic cells such as rabbit reticulocytes (e.g. Pelham (1976), Eur. J. Biochem. Vol. 131, page 289) and wheat germ cells (e.g. Spirin (1990), American Society for Microbiology, 56-70; Stiege (1995), J. Biotechnol. 41:81-90).
As exemplified herein, the addition of RF1 specific, inactivating antibodies to cell free translation systems, preferably to cell free translation systems of prokaryotic origin (e.g. coupled in vitro transcription/translation systems derived from E. coli) leads to dramatic increase of puromycin derivative (e.g. [32P]pGpCpPuromycin or other 5'- modified puromycin derivatives) incorporation into the C-terminus of a protein (e.g. of a full-length esterase), while leaving elongation unaffected. This approach allows the use of (a) conventional coupled transcription/translation system(s) as base for high- yield production of modified proteins, in particular protein-puromycin conjugates. This is, for instance, in contrast to systems where only mRNA without stop codons were employed, leading to very low puromycin analogue incorporation (Nemoto (1999), FEBS Lett., 462, 43-46). Truncation of mRNA makes its 3'-end sensitive to nuclease attack and therefore puromycin is incorporated into a set of C-terminaly truncated proteins. In contrast, transcription from DNA template within the presented system constantly supplies a pool of newly synthesized mRNA with unaffected stop codon protected from 3'-degradation by a downstream ribonucleotide sequence and therefore provides a template for synthesis of a homogenous product. It is demonstrated herein that the inactivation of release factors, in particular of RF1 by corresponding precipitating and/or crosslinking antibodies within the translation mixture allows to use of nonsense-codon suppressing agents, e. g. puromycin, as an addressed delivery vehicle for covalent attachment of different chemical structures to the protein's C-terminus via peptidyl transferase reaction. It is also demonstrated that utilization of biotinylated derivatives of puromycin allows to immobilize the final product on a solid surface, coated e. g. with streptavidin and that the usage of puromycin with attached Cy3 fluorophore resulted in production of a fluorescence- labeled protein (e.g. an esterase). The methodology of the present invention allows the covalent conjugation of (a) chemical structure(s) including different dyes, affinity tags, spin labels etc. on the C-terminus of a protein to be synthesized with high yield by the cell-free translation system provided herein and the methods disclosed in this invention. This opens the way for variety of applications. For example, puromycin modified with an azide group could be used for subsequent one site addressed Staudinger reaction (Kδhn (2004), Angew. Chem. Int. Ed Engl., 43, 3106-3116) and utilization of puromycin carrying α-thio-ester group may allows to use the protein ligation technology (Lovrinovic (2003), Chem. Commun. (Camb. ), 822-823). This conjugation technology can be used to develop concepts for preparation of protein arrays and novel tools to study protein interactions (Ramachandran (2004), Science, 305, 86-90). Puromycin derivatives modified with an azide group may also by used for site-specific protein immobilisation, e.g. by taking advantage of the Staudinger ligation (Soellner, 2003, J.Am.Chem.Soc. 125, 11790-11791.).
Thus, the experiments effected herein demonstrate that in particular puromycin can be used as a delivery carrier of different chemical functional groups for specific C- terminal labelling of protein in high yield. It is further demonstrated that puromycin and corresponding derivatives can be safely used as a nonsense-codon suppressing agent comprised in the cell-free translation system of the present invention.
As further exemplified herein and in analogy to the examples, an addition of anti- release factor antibodies, in particular anti-RF1 specific antibodies to cell-free translation systems, leads to dramatic increase of incorporation of (unnatural) amino acids delivered by suppressor tRNA into a protein. In the appending examples it is demonstrated that a particular protein, namely the Esterase 2 from Alicyclobacillus acidocaldarius (Est2) can be safely and efficiently produced from a template comprising an (artificially) introduced internal stop codon. The coding sequence of said esterase 2 to be employed in terms of the present invention is shown in SEQ ID NO: 38, the corresponding amino acid sequence is shown in SEQ ID NO: 39 or SEQ ID NO. 55. It is preferred that the amino acid sequence of said esterase 2 is that of SEQ ID NO. 55. Within SEQ ID NO: 39, the internal methionine residues (Met (M)), encoded by their corresponding nucleotide residues of SEQ ID NO: 38, are indicated as X. As documented in the examples, incorporation of a serine at amino acid position 155, at the position of the previously introduced STOP-codon, could be obtained. At this position 155, a serine, located in the Ser-His-Asp catalytic triad (Fig. 16B.), is essential for hydrolytic activity (De Simone (2000), J MoI. Biol 303, 761- 771). It is encoded by the ACG triplet at the corresponding position of the est2 mRNA (Hemila (1994), Biochim. Biophys. Acta 1210, 249-253). Accordingly, as exemplified herein below the coding sequence for serine 155 was substituted to a RF1- dependent stop codon (UAG) and the resulting construct was used to test the conditions for efficient termination and/or suppression at UAG stop codon. Thus, it was demonstrated that the impairment of RF1 by precipitating and/or crosslinking antibodies, leads to the situation where the suppressor Ser-tRNASer(CUA) is efficiently bound to the A-site of UAG-programmed ribosomes. This leads to complete suppression of UAG codon and to incorporation of the catalytically essential serine-155 into the enzyme. At high Ser- tRNASer(CUA)ΕF-Tu.GTP concentrations, the competition with other aminoacyl-tRNAΕF-Tu.GTP ternary complexes leads to misreading of near-cognate codons and results in synthesis of error prone or incomplete polypeptide chains void of enzymatic activity. Thus, the use of est2 mRNA(amber 155) as a template and the possibility to deactivate the endogenous RF1 in the in vitro translation system by RF1 antibodies permits an optimal adjustment of RF1 and tRNASer(CUA) concentrations to achieve a complete suppression and at the same time a maximal retention of enzymatic activity of the esterase.
Indeed, substantial rates of misincorporations resulting in inactive enzyme were observed in the present work in the absence of RF1 when the "hungry" UAG stop codon entered the ribosomal A-site or when a very high concentration of one specific aminoacyl-tRNA (in this study tRNASerCUA) was present in the reaction mixture (Fig. 19). This observation has important practical implications and underlines the necessity for determination of optimal and balanced concentrations of amimoacyl- tRNAs and RF1 in order to obtain high yield of active proteins by in vitro translation. As demonstrated herein, the remaining concentration of RF1 in the in vitro translation mixture has to be very low in order to avoid a competition with tRNASer(CUA) (below 10 nM). Under such condition the incorporation of serine from Ser- tRNASer(CUA) into the polypeptide at LJAG codon reaches a full level and opens the possibility to incorporate unnatural amino acids into polypeptide in high yield under full retention of their activity.
The efficiency of UAG suppression by suppressor tRNAs using the translation systems of the prior art, however, did not reach the suppression levels of the present invention. Probably the efficiency of RF1 deactivation in the previously reported attempts was not sufficient to decrease concentrations of active RF1 in the translation mixtures below 10 nM. In particular, use of polyclonal anti-RF1 antibodies (e.g. anti-RF antibodies directed against RF1 of Thermus thermophilus; SEQ ID NO: 2) meets this requirement with excellent results.
A preferred cell-free translation system of the present invention or to be employed in context of this invention is a cell-free coupled transcription/translation system. "Coupled" in context of the present invention means that the transcription and translation occur concurrently in one reaction. "Coupled" in the sense of the present invention can also mean that the mRNA molecules which have just been formed by transcription are already translated by the ribosomes. "Coupled" in context of the present invention can also mean that the transcription and translation occur simultaneously after the addition of DNA to the extract. The use of RNA as a template in E. coli extracts results in protein production but such a reaction is not called "coupled". Further, said cell-free translation system may be of prokaryotic and/or eukaryotic origin, yet preferring it is of prokaryotic origin. For example, and also shown in the experimental part, a cell-free translation system of E. coli origin may be employed. Prokaryotic cell-free translation systems, in particular from E. coli, can be used when the gene to be expressed has been cloned into a vector containing the appropriate prokaryotic regulatory sequences, such as a promoter and ribosome binding site.
The cell-free coupled transcription/translation systems, as employed in context of this invention, may (further) comprise the following ingredients:
- a cell-free extract;
- ribonucleotide triphosphates, like ATP, CTP, GTP, UTP, etc.;
- a RNA polymerase;
- magnesium ions
- a template plasmid; and/or
- amino acids, or a mixture of amino acids to be incorporated in a nascent peptide, polypeptide or peptide, in particular leucine.
Said systems may comprise also aminoacyl-tRNAs as defined herein or as generally known in the art. Said aminoacyl-tRNAs may originally be comprised in the cell-free extract. But also additional tRNAs, like suppressor seryl-tRNASER(CUA) may be added. Preferably the magnesium ions that are contained in the cell-free coupled transcription/translation systems of the invention are at a concentration at which RNA is transcribed from DNA and RNA translates into protein. More preferably, the magnesium ions are in form of MgCI2, e.g. at a concentration of 9-12 mM.
Preferably, the cell-free coupled transcription/translation systems, as employed in context of this invention, may comprise the ingredients as listed below:
- 3OS cell-free extract from E. coli (enzyme- and und ribosomal fraction);
- MgCI2 9-12 mM;
- DTT 10 mM;
- Amino acids, 200 μM each (For labelling, each amino acid can be applied as a 14C amino acid with a concentration of 100 μ M (e.g. 14C-leucine))
- Rifampicin 0,02 mg/ml reaction mixture,
- Bulk-tRNA 600μg/ml reaction mixture,
- ATP,CTP,GTP,UTP, 1mM each,
- Phosphoenolpyruvate 10 mM;
- Acetylphosphate 10 mM;
- Pyruvatekinase 8 μg/ml reaction mixture; - Plasmid 2 pmol/ml reaction mixture;
- T7 Polymerase 500 Units/ml reaction mixture;
- HEPES pH 7,6, 5O mM;
- Potassium acetate70 mM;
- Ammonium chloride 30 mM;
- EDTA pH 8,0 , 0,1 mM;
- Sodium azide 0,02 %;
- Polyethyleneglycol 4000 2 %;
- Protease inhibitors: aprotinin 10 μg/ml reaction mixture, leupeptin 5 μg/ml reaction mixture, pepstatin 5 μg/ml reaction mixture; and
- Folic acid 50 μg/ml reaction mixture.
The above recited cell-free coupled transcription/translation system is merely an illustrative example of a cell-free system to be employed in context of this invention. An inventive cell-free translation system also comprises a) the inactivating anti-RF antibodies and/or sera and b) the nonsense-codon suppressing agent as defined herein. Corresponding examples are also given in the experimental part.
Generally, the composition of cell-free translation systems, in particular cell-free coupled transcription/translation systems is well known in the art. Said systems are also commercially available, e. g. from Promega GmbH or Roche Diagnostics GmbH (Mannheim, Germany). Most preferably, and also shown in the experimental part, said cell-free coupled transcription/translation systems may be comprised in evaluation size transcription/translation kits purchased from RiNA GmbH (Berlin, Germany).
The cell-free translation systems to be employed in context of the present invention may (further) comprise a labelled amino acid. By incorporation of said labelled amino acid, it is possible to monitor and/or track the synthesis of a protein or to identify the location (e.g. in a polyacrylamide gel) of said protein. Preferably, the labelled amino acid is a radioactively labelled amino acid, more preferably the labelled amino acid is [14C]leucine, [14C]valine and/or [14C]isoleucine, most preferably the labelled amino acid is [14C]leucine. The cell-free translation system of the present invention, may also be of eukaryotic origin. In this case, a wheat germ extract cell-free translation system or a rabbit reticulocyte lysate cell-free translation system would be preferred, but a cell-free translation system based on lysates from oocytes or eggs (e.g. oocytes from Xenopus) may be also applicable. These eukaryotic systems may preferably be used for the expression of eukaryotic genes or mRNA and are also well known in the art.
Another preferred cell-free translation system to be employed in the context of this invention is a cell-free translation system, wherein said nonsense-codon suppressing agent is puromycin or a derivative thereof and/or a suppressor aminoacyl-tRNA. Said suppressor tRNA may be, e.g. suppressor seryl-tRNASer (CUA).
The nonsense-codon suppressing agent may also be e.g. selected from the group consisting of:
(a) Puromycin;
(b) 5'-OH-CpPuromycin;
(c) 5'-OH-CpCpPuromycin;
(d) a puromycin derivative as defined in (a) to (c) having a residue covalently attached directly or via a linker to its 5'-position;
(e) a puromycin derivative as defined in (a) to (d) having a residue covalently attached directly or via a linker to the element N4 of the cytosine-residue of an 5' attached cytidine-residue; and
(f) a puromycin derivative as defined in (a) to (e) having a residue covalently attached directly or via a linker to the element C5 of the cytosine-residue of an 51 attached cytidine-residue; whereby a nonsense-codon suppressing agent as defined in (e) is preferred.
For example, the residue to be covalently attached to the puromycin (or a derivate thereof), may be selected from the group consisting of nucleic acids like DNA, RNA, locked DNA, PNA, oligonucleotide-thiophosphates and substituted ribooligonucleotides and other nucleic acids. It is also envisaged that other residues, like peptides or "tags" can be attached to said puromycin to be integrated in a protein during its in vitro synthesis. Further and/or additional modifications on the puromycin structure, in particular of the puromycin-derivatives to be employed and as disclosed herein, are also envisaged. Accordingly, the puromycin (-derivatives) to be employed in context of the present invention and as disclosed herein, may be labelled, preferably at the 5'-End. Said labelling preferably is with a radioactive element, more preferably with 32phosphorus (32P), most preferably with a phosphate group containing 32phosphorus ([32P]p).
In context of the present invention, the term "nucleic acid(s)" and/or "nucleic acid molecule(s)" encompasses all forms of naturally occurring types of nucleic acid(s) and/or nucleic acid molecules as well chemically synthesized nucleic acids and also encompasses nucleic acid analogs and nucleic acid derivatives such as e. g. locked DNA, PNA, oligonucleotide tiophosphates and substituted ribo-oligonucleotides. Furthermore, the term "nucleic acid" and/or "nucleic acid molecules(s)" also refers to any molecule that comprises nucleotides or nucleotide analogs. Preferably, the term "nucleic acid(s)" and/or "nucleic acid molecule(s)" refers to oligonucleotides or polynucleotides, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The "nucleic acids" and/or "nucleic acid molecule(s)" may be made by synthetic chemical methodology known to one of ordinary skill in the art, or by the use of recombinant technology, or may be isolated from natural sources, or by a combination thereof. The DNA and RNA may optionally comprise unnatural nucleotides and may be single or double stranded. "Nucleic acid(s)" and/or "nucleic acid molecule(s)" also refers to sense and anti-sense DNA and RNA, that is, a nucleotide sequence which is complementary to a specific sequence of nucleotides in DNA and/or RNA.
Furthermore, the term "nucleic acid(s)" and/or "nucleic acid molecule(s)" may refer to DNA or RNA or hybrids thereof or any modification thereof that is known in the state of the art (see, e.g., US 5525711 , US 4711955, US 5792608 or EP 302175 for examples of modifications). Such nucleic acid molecule(s) are single- or double- stranded, linear or circular, natural or synthetic, and without any size limitation. For instance, the nucleic acid molecule(s) may be genomic DNA, cDNA, mRNA, antisense RNA, ribozyme or a DNA encoding such RNAs or chimeroplasts (Colestrauss (1996)). Preferably, said nucleic acid molecule(s) is/are in the form of a plasmid or of viral DNA or RNA. Nucleic acid molecuie(s) may also be oligonucleotide(s), wherein any of the state of the art modifications such as phosphothioates or peptide nucleic acids (PNA) are included.
In further examples, the residue, covalently attached to said puromycin or said derivative thereof, may be selected from the group consisting of a Cy3-fluorosphore, biotin or an other affinity tag, a reactive group for affinity labelling or any other reporter group (for review see Gilmore (1999), Topics in Current Chemistry, 202, 77- 99). The reactive group, for instance, can be an azide group to use for subsequent one site addressed Staudinger reaction (Kόhn (2004), Angew. Chem. Int. Ed Engl., 43, 3106-3116) or an α-thio-ester group to use for the protein ligation technology (Lovrinovic (2003), Chem. Commun. (Camb.), 822-823). As mentioned above, puromycin derivatives modified with an azide group may also by used for site-specific protein immobilisation, e.g. by taking advantage of the Staudinger ligation (Soellner, 2003, loc. cit.). In further examples, the residue, covalently attached to said puromycin or said derivative thereof, may be also be a(n) (other) spectroscopic reporter.
In the context of the present invention, the term Jinker" refers to a molecule capable to connect said puromycin (-derivative) and said residue covalently. For example, the linker between the puromycin (-derivative) and said residue may be an aliphatic amine derivative, preferably forming an amide with a fatty acid attached to a polyoxyamine. Preferably, said linker may comprise the following molecule:
Figure imgf000023_0001
or
Figure imgf000023_0002
wherein the part of the molecule indicated in squared brackets may be of different length, e.g. may be elongated by additional or shortened by less carbon residues and/or oxygen residues.
Preferably, in said linker, (n) is at least 3, preferably at least 5 carbon residues. Yet, the amount of carbon residues (n) may most preferably be 5 or 9.
Further, the linker between the puromycin (-derivative) and said residue may act as a
"place holder" that warrants the undisturbed entrance of the puromycin (-derivative) into the A-site of the ribosome.
"Nonsense-codon suppressing agent" are known in the art, as documented above. However, the "nonsense-codon suppressing agent" comprised in a cell-free translation system of the present invention or as described herein may also be selected from the group consisting of:
(a) 5'-OH-GpCpPuromycin;
(b) 5'-OH-GpCpCpPuromycin;
(c) 5'-OH-GpApCpCpPuromycin;
(d) 5'-OH-GpCpApCpCpPuromycin;
(e) 5'-OH-GρCpCpApCpCpPuromycin;
Figure imgf000025_0001
Figure imgf000026_0001
Figure imgf000027_0001
Figure imgf000028_0001
It was surprisingly found that the puromycin derivatives of the present invention or the puromycin derivatives to be employed in the methods provided herein are particularly advantageous for the production of alloproteins, e.g. the alloproteins as defined herein. For example, said puromycin derivatives are especially suitable for the production of alloproteins by conjunction, particularly C-terminal conjugation, of certain compounds to proteins, peptides or polypeptides. Said compounds to be conjuncted with said proteins, peptides or polypeptides may be, but are not limited to, markers or labels, like (oligo-)nucleotides, like, e.g. specific (DNA- or RNA-) probes, reactive groups for affinity labelling, crosslinking or attachment to macromolecular or solid surfaces and vesicles, like e.g. (oligo-)saccharides, affinity ligands like e.g. lipids, spectroscopic labels for UV/VIS- and fluorescence-spectroscopies including single molecule and fluorescence energy transfer spectroscopy, spin labels and metals, metal complexes and metal clusters. Furthermore the puromycin derivatives of the present invention or the puromycin-derivatives to be employed in the methods provided herein are particularly advantageous for the immobilisation of, e.g., proteins, peptides or polypeptides onto certain surfaces, like, e.g., glas surfaces, sepharose surfaces or polymeric surfaces, like, e.g. polystyrene surfaces. Said surfaces may be covered by further substances, like, e.g. streptavidin, biotin or polylysine. For the conjunctions with the mentioned compounds or the mentioned immobilisation, puromycin derivatives carrying an azide group, e.g. the puromycin derivative indicated under (i), above, are particularly preferred. However, this preferred puromycin derivatives may also be employed in further aplications. Moreover, in order to achieve covalent conjugations, a person skilled in the art is not limited to this type of chemistry. Furthermore, a person skilled in the art is aware of further compounds to be conjuncted with proteins, peptides or polypeptides. As already pointed out above, the cell-free translation system described herein and to be employed in the context of this invention is a cell-free translation system, wherein said release factor contained and to be specifically inactivated by precipitation and/or crosslinking, whereas all other components remain intact, is of prokaryotic or eukaryotic origin, more preferable it is of prokaryotic origin, even more preferably it is from E.coli. For example, a release factor to be inactivated from E. coli may be the release factor 1 , the release factor 2, the release factor homolog 1 , the release factor homolog 2, the release factor homolog 3 or the release factor homolog 4. Said release factors may be encoded by the nucleotide sequences as shown in SEQ ID NOs: 5, 7, 9, 11 or 13 and/or may have the amino acid sequences as shown in SEQ ID NOs: 6, 8, 10, 12, 14 or 37. Most preferred, and also shown in the experimental part, said release factor contained in said cell-free translation system and to be inaktivated is the release factor 1 from E. coli. Said most preferred release factor may be encoded by the nucleotide sequence as shown in SEQ ID NO: 5 and/or may have the amino acid sequence as shown in SEQ ID NO: 6.
The release factor contained and to be inactivated in the cell-free translation system of the present invention may be different from the release factor, against which the antibody to be employed was directed and/or generated. For example, the release factor contained and to be inactivated in the cell-free translation system of the present invention may be from E. coli. Accordingly, sad translation system comprises RF1 from E. coli. Yet, as shown in the examples, the anti-release factor antibody, precipitating and/or crosslinking said release factor, was generated against a release factor from Thermus thermophilus, namely against RF1 from Thermus thermophilus. Said RF1 from Thermus thermophilus may be encoded by the nucleotide sequence as shown in SEQ ID NO: 1 and/or may have the amino acid sequence as shown in SEQ ID NO: 2.
In an eukaryotic context, the release factor to be inactivated by a specific crosslinking and/or precipitating antibody may be from rabbit, fruit fly or yeast. Preferably, said release factor to be inactivated by antibodies is a rabbit RF.For instance, said release factor is the release factor 1 or the release factor 3 from rabbit, release factor 1 from fruit fly or the release factor 1 or the peptide chain release factor 1 from yeast. Said exemplified release factors may be encoded by the nucleotide sequences as shown in SEQ ID NOs: 15, 17, 19 or 21 , respectively, and/or may have the corresponding amino acid sequences as shown in SEQ ID NOs: 16, 18, 20 or 22, respectively. Corresponding antibodies may be prepared by methods known in the art, for example by the generation of a polyclonal serum against said release factors. "inactivating antibodies to be employed in the cell-free translation system of the present invention are, as described herein, antibodies and/or antibody molecules which are capable of precipitating and or crosslinking the release factor(s) comprised in the cell-free translation system of the present invention. Said "inactivation" may be a complete or a partial inactivation. As pointed out above, said "inactivation" leads to an inactivation of the function of said release-factors of at least 60%, more preferably of at least 70%, more preferably of at least 80% and more preferably of at least 90%. The corresponding inactivation of the release-factors by the addition of the precipitating and/or croslinking antibodies and/or antibody molecules can be measured by methods known in the art, as already mentioned above.
In particular, the release factor contained in said cell-free translation system and to be inactivated may be selected from the group consisting of:
(a) a release factor encoded by a nucleotide sequence comprising a nucleotide sequence as shown in any one of SEQ ID NOS: 1; 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21, 23, 25, 27, 29, 31 , 33 and 35;
(b) a release factor encoded by a nucleotide sequence coding for a polypeptide comprising an amino acid sequence as shown in any one of SEQ ID NOS: 2; 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 and 37;
(c) a release factor which is encoded by a nucleotide sequence of a nucleic acid molecule that hybridizes to the complement strand of a nucleic acid molecule comprising a nucleotide sequence as defined in (a) or (b) and which releases a translation product from a ribosome in a cell-free translation system;
(d) a release factor which comprises an amino acid sequence as shown in any one of SEQ ID NOS: 2; 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 and 37;
(e) a release factor which comprises an amino acid sequence which is at least 40% identical to the full length amino acid sequence as shown in any one of SEQ ID NOS: 2; 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 and 37; and
(f) a release factor encoded by a nucleotide sequence which is degenerated to a nucleotide sequence as defined in any one of (a) to (c).
In the context of the present invention the term "hybridizes" refers to hybridization under conventional hybridization conditions, preferably under stringent conditions, as for instance described in Sambrook and Russell (2001), Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor, NY, USA. In an especially preferred embodiment, the term "hybridizes" refers to hybridization that occurs under the following conditions: Hybridization buffer: 2 x SSC; 10 x Denhardt solution (Fikoll 400 + PEG + BSA; ratio 1 : 1: 1); 0.1 % SDS; 5 mM EDTA; 50 mM Na2HP04; 250, ug/ml of herring sperm DNA; 50 ug/ml of tRNA; or 0.25 M of sodium phosphate buffer, pH 7.2; 1 mM EDTA 7% SDS Hybridization temperature T = 60 °C Washing buffer: 2 x SSC; 0. 1 % SDS Washing temperature T = 60°C. Polynucleotides which hybridize to the complement strand of a nucleic acid molecule, comprising a nucleotide sequence as defined herein, can, in principle, encode a polypeptide having release factor activity from any organism expressing such polypeptides or can encode modified versions thereof. Polynucleotides which hybridize with the polynucleotides as defined in connection with the invention can for instance be isolated from genomic libraries or cDNA libraries of bacteria, fungi, plants or animals. Preferably, such polynucleotides are of procaryotic origin, particularly preferred from Thermus thermophilυs or E. coli. Furthermore, the release factor contained in said cell-free translation system may also be least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, even more preferably at least 90% and most preferably at least 95% identical to the full length amino acid sequences as shown in any one of SEQ ID NO: 2; 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 and 37.
The cell-free translation system of the present invention is a cell-free translation system, wherein said precipitating and/or crosslinking anti-RF antibody is directed against a release factor as defined herein-above. Preferably, as documented in the examples, a crosslinking and/or precipitating antibody against the RF1 from Thermus thermophilus is employed in particular in cell-free translation/transcription systems of prokaryotic origin (i.e. in E. coli cell- free systems). Said anti-RF antibody may also be monoclonal or polyclonal, preferably polyclonal. Said anti-RF antibody may be purified. Preferably, said anti-RF antibody is provided in a serum. It was surprisingly found, that (an) antibody molecule(s) and/or serum directed against RF1 from Thermus thermophilus is particularly useful in precipitating and/or crosslinking (and thereby inactivating) the RF1 of E. coli in a prokaryotic translation system derived from E. coli.
Accordingly, in a most preferred embodiment of the cell-free translation system of the present invention, said cell-free translation system is an E. coli cell-free translation system, said anti-RF antibody precipitating and/or crosslinking a release factor contained in said cell-free translation system is an antibody directed against the release factor 1 from Thermus thermophilus. The release factor contained and to be inactivated in said cell-free translation system is release factor 1 (RF1) from E. coli.
Even if antibodies and/or polyclonal sera directed against RF1 of Thermus thermophilus are to be employed, in a preferred embodiment of the invention, in E. coli derived translation systems, also antibodies and/or sera and/or antibody molecules specifically directed against E. coli RF1 may be employed. Antibodies against RF1 from E. coli possess the same RF1 deactivation properties as a precipitating and/or crosslinking antibody against RF1 of Thermus thermophilus . Yet, the precipitating and/or crosslinking sera specific for RF1 of E. coli and generated against RF1 of E. coli have to be added at higher concentration. The reason may be that the amount of anti- RF antibody molecules, obtained by immunization of a non- human vertebrate, is lower in serum obtained by immunization with RF1 from E. coli than in serum obtained by immunization with RF1 from Thermus thermophilus. However, the dilution of the translation mixture caused by increasing amount of sera and increasing amount of contaminating material may be the cause of translation inhibition. This, in some cases, may restrain the use of "anti-E. coli RF" protein antiserum in translation systems derived from E. coli. Accordingly, the use of an antibody and/or serum against RF1 from Thermus thermophilus, is more preferred, in particular in translation systems derived from E. coli. The same amount of added antibodies result in approximately the same incorporation of puromycin derivative. Without being bound by theory, immunization of rabbits with the T. thermophilus RF1 may lead to generation of higher titer of antibodies.
Again, without being bound by theory, environmental temperature may be important in the generation of valuable antibodies against RFs, preferably antibodies against RF1. Indeed the protein from termophilic organism living at 65°C should be conformationally "frozen" in rabbit blood (37°C) and therefore be more homogenic matrix for antibody formation. In contrast, E. coli protein is under same conditions as in nature and can possess conformation flexibility.
The person skilled in the art is readily in the position to deduce the amount of anti- release factor antibody to be added to the cell-free system. Corresponding embodiments are given in the appended examples. Normally, said anti-release factor antibody/antibody molecule is added in an amount that the de-activation/inhibition of the given release factor reaches an plateau.
Furthermore, the present invention relates to a kit comprising the cell free translation system of the present invention. Preferably said kit comprises individual ingredients of the cell-free system and in particular the two essential further components of the inventive cell-free systems, namely an anti-RF antibody to be added to the cell-free translationsystem and nonsense-codon suppressing agent(s). Preferably, the cell- free translation system, the antibody (antibody preparation and/or serum) and the nonsense-codon suppressing agent(s) are individually packed in said kit, preferably in individual vials. However, it is also envisaged that all components of the invention are already comprised in one vial.
As discussed above, a particular preferred antibody and/or serum to be used in the cell-free translation system of the present invention is an antibody and/or serum directed and or generated against RF1 of Thermus thermophilus. Said antibody and/or serum is particulary preferred in prokaryotic cell-free translation systems, e.g. translation systems derived from E. coli. It was surprisingly found, that generated against RF1 of Thermus thermophilus are capable of efficiently inhibiting and/or inactivating and/or deactivating the function of E. coli RF1. Accordingly, the present invention also relates to an anti-RF antibody obtainable by
(a) eliciting an in vivo humoral response against release factor 1 from Thermus thermophilus or a fragment thereof in a non-human vertebrate; and
(b) obtaining a serum from said non-human vertebrate.
The term "anti-RF antibody", in particular "anti-RF1 antibody" as employed in context of the present invention refers to an antibody, a plurality of antibodies and/or a serum comprising such antibodies which is/are able to specifically bind to, interact with, detect and/or precipitate and/or crosslink RFs, in particular RF1 , preferably RF1 from E. coli or a fragment thereof. Said term also relates to a purified serum, i.e. a purified polyclonal serum. The antibody molecule is preferably a full immunoglobulin, like an IgG, IgA, IgM, IgD, IgE, IgY (for example in yolk derived antibodies). The term "antibody" as used in this context of this invention also relates to a mixture of individual immunoglobulins. Furthermore, it is envisaged that the antibody/antibody molecule is a fragment of an antibody, like an F(ab), F(abc), Fv Fab' or F(ab)2. Furthermore, the term "antibody" as employed in the invention also relates to derivatives of the antibodies which display the same specificity as the described antibodies. Such derivatives may, inter alia, comprise chimeric antibodies or single- chain constructs. Yet, most preferably, said "anti-RF(1) antibody" relates to a serum, more preferably a purified (polyclonal) serum and most preferably to a crude polyclonal serum. The antibody/serum is obtainable, and preferably obtained, by the method described herein and illustrated in the appended examples. The term "eliciting an in vivo humoral response in a non-human vertebrate" relates to the provocation of an immune response in a non-human vertebrate, in particular the provocation of an antibody response to RF1 from Thermus thermophilus or a fragment thereof. Said antibody response comprises primary as well as secondary antibody responses to the antigenic challenge with RF1 from Thermus thermophilus or a fragment thereof. The term "eliciting an in vivo humoral response", accordingly, relates to the provocation of an immune reaction involving the production of antibodies directed towards the antigen, namely RF1 from Thermus thermophilus or a fragment thereof. Said release factor 1 from Thermus thermophilus or said fragment thereof may be naturally occurring release factor 1 or it may, preferably, be recombinantly produced. Naturally release factor 1 may be purified by methods known in the art and also the recombinantly produced RF1 may be further purified before a non-human vertebrate is immunisized with said RF1 or an RF1 comprising preparation. Accordingly, said release factor 1 from Thermus thermophilus or said fragment thereof may be purified. The term "RF 1 from Thermus thermophilus or a said fragment thereof is purified" relates preferably to an isolated RF1 from Thermus thermophilus protein or fragment thereof, which has been purified to homogeneity. The coding sequence and/or the protein sequence of RF1 of Thermus thermophilus is known in the art; see also SEQ ID No. 1 and 2. In particular, it has been purified to a purity level of at least 80%, more preferably of at least 85%, even more preferably of at least 90%, particularly preferred of at least 95% purity. The purity of RF1 from Thermus thermophilus protein may be confirmed by methods known in the art. Accordingly, the purified preparation of RF1 from Thermus thermophilus to be employed in the immunization protocols described herein comprises, preferably, less than 5% contaminating, unrelated proteins or protein fragments. Most preferably, said preparation comprises less than 2% contaminating, unrelated proteins or protein fragments. Corresponding examples for a purification are given in the appended examples. Purity of the purified RF1- preparation may be measured by methods known in the art which comprise gel stainings (in particular silver stains of SDS-PAGE followed by densitometric analysis) NMR-measurements or mass spectroscopy (MS), in particular, MALDI mass spectroscopy. As illustrated in the appended example, preferably, said RF1 from Thermus thermophilus or a fragment thereof may be purified by ion exchange chromatography. Said purification may further comprise a gel filtration. Said purification may further comprise, prior to ion exchange chromatography, a protein precipitation step, preferably an AMS-precipitation step. Said purification may further comprise, after the ion exchange chromatography a hydroxyapatite chromatography step and/or an additional precipitation step and/or additional ion exchange chromatography step and/or additional ammonium-sulfate-precipitation step. More preferred, said purification may further comprise, most preferred after the first ion exchange chromatography, a heat treatment. Said heat treatment is preferred. For example and more preferred, said heat treatment may be for 15 minutes at 65 °C. Ion exchange chromatography is known to the artisan and ion exchange media comprise, but are not limited to Mini beads Q, Source 15 Q, Source 30 Q, Sepharose High Performance Q, Sepharose Fast Flow Q1 Sepharose XL Q, Sepharose Big Beads Q, DEAE, Streamline DEAE (all from Amersham Biosciences, Vienna, Austria), DEAE-cellulose, QA-cellulose, CM-cellulose, SE-cellulose, DE-52 (Whatman, Kent, England) or Agarose based ion exchangers. Most preferably a Q- Sepharose FF column (Amersham Biosciences, Vienna, Austria) or an EMD-SO3 column (Merck) is employed. It is of note that also normal gravity flow or FPLC systems may be employed.
Gel filtration systems and media are also known to the skilled artisan which comprise Superdex peptide, Superdex 30, Superdex 200, Superose 6, Superose 12, Sephacryl, Sphadex, Biogel P, Agarose-gel, Fracto-gel or Ultro-gel.
Protein precipitation techniques comprise, inter alia, Dextran sulphate-, Polyethylene glycol (PEG) 4000 - 8000-, Acetone-, Protamne sulphate-, Streptomycin sulphate-, pH-shift-precipitations. Preferably, said protein precipitation is carried out by ammonium sulfate precipitation as known in the art.
A preferred anti-RF antibody of the present invention, as obtained in the eliciting step (a) of the above recited method may be purified or may be provided in a serum. Said non-human vertebrate, from which said serum containing said anti-RF antibody and/or said anti-RF antibody is obtained, may be selected from the group consisting of rat, mouse, rabbit, chicken, sheep, horse, goat, pig and donkey. Preferably, said non-human vertebrate is rabbit.
The anti-RF antibody of the present invention may also obtainable by eliciting an in vivo humoral response against a native release factor from Thermus thermophilus or a fragment thereof in a non-human vertebrate. For example, the (purified) RF1 from Thermus thermophilus may be a native, RF1 from Thermus thermophilus as defined herein. It is preferred that said RF1 from Thermus thermophilus is a full length protein, comprising preferably 354 amino acids. The antibodies of the present invention provide for the first time a reliable tool to (exhaustively) precipitate and/or crosslink RFs and thereby (exhaustively) deplete said RF's from e.g. cell-free translation systems. It was surprisingly found that the anti-RF1 antibodies produced according to the above-described method are, in contrast to e.g. aptamers against RFs, capable of reliably depleting RFs, in particular RF1 from E. coli from cell-free translation systems.
The present invention also relates to the use of an anti-RF antibody as defined herein-above and/or the anti-RF antibody and/or serum of the present invention for the preparation of a cell-free translation system.
In a preferred embodiment of the invention a method for the production of an anti-RF antibody is provided, said method comprising the steps of
(a) eliciting an in vivo humoral response against release factor 1 from Thermus thermophilus or a fragment thereof in a non-human vertebrate; and
(b) obtaining a serum from said non-human vertebrate.
As described above, said release factor 1 from Thermus thermophilus or said fragment thereof may be purified. Likewise, said anti-RF antibody as obtained in the eliciting-step (a) may be purified. For example, said release factor 1 from Thermus thermophilus or said fragment thereof is a native-release factor from Thermus thermophilus or a fragment thereof. Preferably, the RF1 from Thermus thermophilus has the amino acid sequence as shown in SEQ ID No. 2 or is encoded by a nucleic acid molecule as shown in SEQ ID No. 1. The person skilled in the art is aware of the fact that the term "RF1 of Thermus thermophilus" also comprises variants and derivatives of said RF1 from Thermus thermophilus. Preferably, said variants and derivatives comprise at least 80% identity in amino acid sequence to the Thermus thermophilus-RFI sequence as shown in SEQ ID No. 2. it is of note that for the preparation of a polyclonal serum directed against RF1 of Thermus thermophilus also fragments, in particular immunogenic fragments of said amino acid sequence as shown in SEQ ID No. 2 may be employed. Said fragments, preferably, comprise at least 10, more preferably at least 12, more preferably at least 15 amino acid residues. For immunization practice, said fragments may be linked to bulk proteins, like KHL in order to facilitate the immunization in non-human vertebrates. Corresponding methods are well known in the art and, inter alia, described in Harlow/Lane ("Antibodies: A laboratory manual" (1988), CSHL, New York).
The present invention also relates to a method for the production of alloproteins, comprising the step of translating RNA into translation product in a cell-free translation system as disclosed herein.
In context of the present invention, the term "alloproteins" refers to proteins that are achieved by applying the subject-matter of the present invention. Said term also refers to proteins having covalently bound a non-proteinaceous molecule which usually is not part of (the )naturally occurring protein(s). Said alloprotein may, for example, comprise a puromycin and/or derivative thereof as defined herein. Furthermore, said proteinaceous molecule may comprise an unnatural amino acid (for examples see, Gilmore (1999), Topics in Current Chemistry, 202, 77-99). Furthermore, said molecule being covalently bound to and comprised in the alloprotein, might be a functional substituent. Various functional substituents of proteins are well-known in the art. For instance, these functional substituents may be oligosaccharides, lipids, fatty acids, phosphates, acetates or other functional groups to modify polypeptide chains of functional proteins (e. g. Eisele (1999), Bioorganic and Medicinal Chemistry 7,193-224).
Furthermore, said molecule might be a residue of a puromycin (-derivative) as defined herein and/or a puromycin (derivative) as defined herein itself and/or the puromycin derivative of the present invention itself.
The alloproteins produced by the method of the present invention, may be used in a wide variety of applications, for example the preparation of synthetic enzymes (Corey (1987), Science, 238, 1401-1403), gene therapy (Zanta (1999), Proc. Natl. Acad. Sci. U.S.A., 96, 91-96), construction of protein microarray (Niemeyer (1994), Nucleic Acid Res., 22, 5530-5539), creation of molecular scale devices (Keren (2002), Science, 297, 72-75), and development of immunological assays (Niemeyer (2003), Nucleic Acids Res., 31, e90). The method for the production of alloproteins, as provided herein, offers the possibility that any desired chemical structure including different dyes, affinity tags, spin labels etc. may be covalently conjugated with proteins at a high yield. This opens the way for variety of applications. For example, puromycin modified with an azide group may be used to covalently attach to the C-terminus of proteins for subsequent one site addressed Staudinger reaction (Kohn (2004), Angew. Chem. Int. Ed Engl., 43, 3106-3116) and utilization of puromycin carrying α-thio-ester group may allow to use the protein ligation technology (Lovrinovic (2003), Chem. Commun. (Camb. ), 822-823). This conjugation technology can further be used to develop concepts for preparation of protein arrays and novel tools to study protein interactions (Ramachandran (2004), Science, 305, 86-90). As mentioned above, puromycin derivatives modified with an azide group may also by used for site-specific protein immobilisation, e.g. by taking advantage of the Staudinger ligation (Soellner, 2003, loc. cit.).
In a preferred method for the production of alloproteins as provided by the present invention, said alloproteins may comprise, as proteinaceous part, proteins selected from the group consisting of enzymes, growth factors, cytokines, toxins, hormones, pheromones, structural proteins and the like. It is also envisaged that said alloproteins only comprise fragments, like functional, active fragments of said enzymes, hormones, pheromones, growth factors, cytokines, toxins, structural proteins and the like. Said proteins may act as act as core-proteins and/or starting proteins for the alloproteins to be produced in the cell-free translation system of the present invention and corresponding additional chemical structures may be added to said proteinaceous part.
Said alloproteins may also be conjugates of proteins and nucleic acids, preferably nucleic acids having (a) specific sequence(s). Said conjugates allow to link the properties of these two distinct groups of biopolymers within one molecule. Therefore, said conjugates can be used in a wide variety of applications, where said linkage of said properties of these two distinct groups of biopolymers is advantageous. Applications of alloproteins as described herein are well known in the art and may, for instance, include the preparation of synthetic enzymes (Corey (1987), Science, 238, 1401-1403), gene therapy (Zanta (1999), Proc. Natl. Acad. Sci. U.S.A., 96, 91- 96), construction of protein microarray (Niemeyer (1994), Nucleic Acid Res., 22, 5530-5539), creation of molecular scale devices (Keren (2002), Science, 297, 72-75), and development of immunological assays (Niemeyer (2003), Nucleic Acids Res., 31 , e90).
As an example also provided in the experimental part of this invention, the alloproteins or product produced by the method of the present invention, may comprise esterases. In context of the present invention, the term "esterases" refers to a molecule which catalyzes the cleavage of an ester into an alcohol and an carboxylic acid. In context of the present invention, the term "alcohol" refers to a compound carrying at least one hydroxyl group and the term "carboxylic acid" refers to a compound carrying at least one carboxyl group. More preferably, and also shown in the experimental part, said alloproteins may comprise the esterase 2 from Alicyclobacillus acidocaldarius (Manco, G. (1998), Biochem. J 332 (Pt 1), 203-212). The sequences coding for Est2 are known in the art and, e.g. obtainable from Hemila (1994), Biochim. Biophys Acta 1210, 249-253. Furthermore, said sequences are documented herein under SEQ ID. No. 38 (coding sequence) and by the expressed amino acid sequences shown in SEQ ID NO. 39 or SEQ ID NO: 55. It is evident that the person skilled in the art may modify said sequences for specific purposes. For example, as also done herein, specific further/additional restriction sites may be introduced. A corresponding example is given in the Est2 sequence comprised in a plasmid provided herein and shown in Fig. 3 (SEQ ID NO: 40). The person skilled in the art is readily in the position to understand that the embodiments provided herein are easily transferable to other proteins, peptides or polypeptides. The person skilled in the art can, e.g. replace the "esterase", as employed as "marker" in the appended examples by any desired protein, peptide or polypeptide, without deferring from the gist of the present invention. Accordingly, for example a growth factor (or any other protein) may be produced which comprises at least, e.g. one additional unnatural amino acid or (e.g.) a puromycin-derivative as defined herein. The Esterase (Est2) from Alicyclobacillus acidocaldarius (SEQ ID NO: 39 or 55) is a thermostable enzyme that consists of one polypeptide chain and possesses a broad substrate specificity (Manco, G. (1998) Biochem. J 332 ( Pt 1), 203-212). Due to high thermostability, practically instant folding and refolding and easily detectable activity, this esterase has a potential application as a reporter for in vitro and in vivo protein expression systems. The tertiary structure of the esterase was determined by X-ray crystallography (De Simone, G. (2000) J MoI. Biol 303, 761-771). Serine 155, located in the Ser-His-Asp catalytic triad (Fig. 16B.), is essential for hydrolytic activity (De Simone, G. (2000) J MoI. Biol 303, 761-771) It is encoded by the ACG triplet at the corresponding position of the est2 mRNA (Hemila (1994), Biochim. Biophys. Acta 1210, 249-253). As already mentioned before and exemplified herein below, the coding sequence for serine 155 was substituted to a RF1 -dependent stop codon (UAG) and the resulting construct was used to test the conditions for efficient termination and/or suppression at UAG stop codon.
Many investigators dealing with mechanism of termination and suppression of termination codons used SDS-PAGE as a criterion for monitoring of suppression events. The possibility of increased translation error rates due to high concentrations of unnatural suppressor tRNAs were usually disregarded. The construction of Est2 mRNA(amber 155) from the template pEst2_amber 155 (see herein below) allows to monitor in parallel the efficiency of the UAG suppression by a band shift in SDS- PAGE and the accumulation of esterase activity in the in vitro translation mixture. This assay is suitable for estimation of optimal conditions to achieve highly efficient suppression in different in vitro translation mixtures. Such assessment seems to be very important since translation systems may individually differ from each other due to different source and preparation method.
In another preferred method for the production of alloproteins as provided herein, said alloproteins are proteins that have covalently attached puromycins or and/a derivative thereof. Furthermore, said alloproteins may have incorporated an (unnatural) amino acid delivered by suppressor aminoacyl-tRNA. Said suppressor aminoacyl-tRNA may be e.g. suppressor serine-tRNASer(CVA) Corresponding additional residues, covalently attached to said puromycin or said derivative thereof, have been described herein above. Also the linker between the puromycin (-derivative) and said residue have been described above and are employed in the context here, mutatis mutantis. Most preferably in the herein provided method for the production of alloproteins, said alloproteins are proteins which have, at their C-terminus a puromycin or a corresponding derivative covalently attached.
Therefore, the present invention also provides for synthetic puromycin derivatives which are particulary useful in context of the preparation of alloproteins. Said alloproteins are preferably prepared using the method disclosed herein and employing the cell-free translation system of the present invention.
The present invention also provides for a puromycin derivative having a residue covalently attached directly or via a linker to one of its elements selected from the group consisting of:
(a) the element N4 of the cytosine-residue of an 5' attached cytidine- residue; and
(b) the element C5 of the cytosine-residue of an 5' attached cytidine- residue; whereby the element as defined in (a) is preferred.
The embodiments described above in context of further residues attached to the puromycin or puromycin derivative, as well as the embodiments described in context of corresponding "linkers" and "linker structures" apply here, mutatis mutandis. Preferably, the puromycin derivatives described herein are to be employed in the generation of alloproteins. Said derivatives may be selected from the group consisting of:
Figure imgf000044_0001
44
Figure imgf000045_0001
Figure imgf000046_0001
Figure imgf000047_0001
The puromycin derivative of the present invention or the puromycin-derivative to be employed in methods provided herein may be particularly useful in the cell-free translation system, the kit comprising said cell-free translation system, the method for the production of alloproteins. For example, the puromycin derivative of the present invention may be useful in mRNA display. The yields of the mRNA-protein coupling in mRNA display (Roberts, (1997) JW Proc Natl. Acad. Sci. U.S.A. 94, 122297-302) are usually low. The reason is the low tolerance of the ribosomal A-site for 5'-extended puromycin-nucleic acid conjugates and the high selectivity of this site for EF-Tu. GTP dependent delivery of the aminoycyl-tRNA (Starck (2002), RNA, 8 890-903) RNA molecules that are longer than 5-6 nucleoitde residues can not enter the ribosomal A- site in EF-Tu. GTP independent manner (Fig. 11). There is, however, a possibility to circumvent this problem by using the puromycin derivative of the present invention. By the provision of said puromycin derivative, instead of covalent attachment of RNA to the 5'-position of puromycin an alternative strategy by which the RNA (mRNA) or other functional groups are attached directly or via a linker to the nculeobases of puromycine-derived olignucleotides (e.g. CpCpPu or CpPu) can be used. Example for this type of conjugation is demonstrated in Fig. 5.
Accordingly, the present invention also provides for the use of a puromycin derivative having a residue covalently attached directly or via a linker to one of its elements selected from the group consisting of:
(a) the element N4 of the cytosine-residue of an 5' attached cytidine- residue; and
(b) the element C5 of the cytosine-residue of an 5' attached cytidine- residue; in a cell-free translation system as defined herein above, in a method for the production of alloproteins as defined herein above and/or in a kit as defined herein above; In all embodiments the element as defined in (a) is preferred.
The potential residues of said puromycin derivative have been described above and the corresponding embodiments apply also here in context of the here disclosed advantageous puromycin attachment sites. Likewise, the linkers between the puromycin (-derivative) and the residues which may, inter alia be employed have been described above in context of other embodiments.
In a preferred embodiment of the herein provided use of a puromycin derivative, the residue is selected from the group consisting of a Cy3-fluorosphore, biotin or another affinity tag, a reactive group for affinity labelling or any other reporter group. Further, the residue may be a(n) (other) spectroscopic reporter.
It is evident for the skilled artesian that other residues may be employed in context of this invention.
In a further preferred embodiment of the herein provided use of a puromycin derivative, the linker is an aliphatic amine derivative, in particular an aliphatic amine forming an amide with a fatty acid as described above.
The puromycin derivative disclosed herein may be selected from the group consisting of:
Figure imgf000050_0001
Figure imgf000051_0001
Figure imgf000052_0001
Figure imgf000053_0001
These and other embodiments are disclosed and encompassed by the description and examples of the present invention. Further literature concerning any one of the compounds, kits, methods and uses to be employed in accordance with the present invention may be retrieved from public libraries, using for example electronic devices. For example the public database "Medline" may be utilized which is available on the Internet, for example under http://www.ncbi.nlm.nih.gov/PubMed/medline.html. Further databases and addresses, such as http://www.ncbi.nlm.nih.gov/, http://www.infobiogen.fr/, http://www.fmi. ch/biology/research_tools. html, http://www.tigr.org/, are known to the person skilled in the art and can also be obtained using, e.g., http://www.lycos.com. An overview of patent information in biotechnology and a survey of relevant sources of patent information useful for retrospective searching and for current awareness is given in Berks, TIBTECH 12 (1994), 352-364.
The present invention is further described by reference to the following non-limiting figures and examples.
The Figures show:
Figure 1.
A: Biosynthesis of proteins in all living organisms is based on peptidyl transferase reaction catalyzed by the ribosome. A nucleophilic attack on the carbonyl group of the ester bond of the peptidyl-tRNA by the amino group of the aminoacyl-tRNA results in formation of an amide (peptide) bond.
B: At concentrations greater than 10 μM the antibiotic puromycin, that by its structure resembles the aminoacylated 3'-terminal adenosine of the aminoacyl-tRNA, can compete with aminoacyl-tRNA.EF-Tu.GTP ternary complex for the ribosomal A-site. In such case it serves as a low molecular weight acceptor substrate in peptidyl transferase reaction leading to formation of puromycin-peptide that is released from ribosomes (Pavlov (1998), J MoI. Biol 284, 579-590).
Figure 2. Plasmid map of pEst2 (plVEX 2.3d-Est2) with terminal amber stop codon.
Figure 3.
Nucleotide sequence of plVEX 2.3d-Est2 (pEst2; SEQ ID NO: 40). Bolded letters are the coding sequence (SEQ ID NO: 38) of esterase 2 (SEQ ID NO: 39 or 55).
Figure 4.
Puromycin derivatives used for the synthesis of conjugates with esterase 2 within the present application or for incorporation into other proteins, peptide or polypeptides to produce alloproteins. The puromycin derivatives were radioactively labelled or can be radioactively labelled as described herein (Example 8).
Figure 5.
A: Structure of CpPu modified on the cytosine residue (4 (N4)) by a linker carrying a functional group (R= oligonucleotide, affinity label, spectroscopic label, biotin, Cy3, azide group, hydrogen residue or other residue, either with or without an additional linker). Alternative positions for attachment of functional groups directly or via a linker are indicated by numbers (5, 6, 8).
B: Structure of CpPu as in A, wherein the part of the molecule indicated in squared brackets is of different length, e.g. is elongated by additional or shortened by less carbon residues and/or oxygen residues.
Figure 6.
C-terminal incorporation of [32P]-GpCpPuromycin into polypeptides during in vitro transcription/translation of plVEX 2.3d-Est2 (pEst2) in the presence of rabbit serum antibodies against RF1 from Thermus thermophilus.
A: Radioactive image of SDS-PAGE of 2 μL samples withdrawn from translation mixture after 90 min. of incubation in the presence of 6 μM [32P]GpCpPuromycin and different amounts of antibodies against RF1 from Thermus thermophilus, Lane 1 , no antibodies added. In lanes 2, 3, 4, 5, 6, 7, 8, 9 the rabbit serum anti-RF1 antibodies were diluted 1 :3000, 1 :300, 1 :150, 1:75, 1:40, 1 :30, 1 :15 and 1:7.5, respectively. Position of the full-length esterase is indicated by arrow. B: The dependence of the reaction yield upon added amount of anti-RF1 antibodies. The reaction yield was determined as a ratio between pmols of [32P]GpCpPuromycin incorporated into the full-length esterase and pmols of the full-length synthesized esterase (the initial values were calculated from corresponding bands on radioactive images).
Figure 7.
Effect of antibodies against Thermus thermophilus RF1 on the incorporation of
[32P]PupCpCp into the esterase during in vitro translation.
In-vitro translation was carried out in the presence of different amounts of antibodies against RF1. Lane 1 , no antibodies added, lanes 2, 3, 4, 5, 6, 7, 8, adition of 0.01,
0.1, 0.2, 0.4, 0.8, 2.0, and 4 μl serum antibodies per 30 μl translation mixture, respectively.
Figure 8.
Comparison between the ability of aptamers and antibodies against Thermus thermophilus release factor 1 to enhance C-terminal incorporation of Pu-derivatives into esterase 2.
A: Efficiency of C-terminal incorporation of [32P]CC-puromycin in the presence of active RF1 (complete translation system without antibody against RF1) during transcription/translation of pEst2. Incubation time is indicated above the corresponding lane.
Lane 1: no puromycin derivative added Lane 2: 1 μM [32P]p-CC-puromycin (42 μM; 301 mCi/pmol) Lane 3: 5 μM [32P]p-CC-puromycin (42 μM; 301 mCi/pmol) Lane 4: 10 μM [32P]p-CC-puromycin (42 μM; 301 mCi/pmol) The arrows indicate the position of the esterase.
B: Influence of DNA encoded anti-RF1 aptamers (aptamers RNA (50 nucleotide long RNA Oligonucleotides with strongest affinity to RF1 from E. coli (see, Szkaradkiewicz (2002), FEBS Lett. 514, 90-95)) were produced by T7 transcription within the reaction mixture) on C-terminal incorporation of CC-Puromycin in the cell-free translation system programmed by pEst2. 2.5 μM [32P]p-CC-puromycin (301 mCi/pmol) was used for in-vitro translation together with 500 nM PCR produced DNA fragments containing sequence coding for RNA Aptamer, 2 nM pEst2.
Lane 1 : no aptamer, Lane 2: presence of aptamer #3, Lane 3: Presence of aptamer
#23.
The arrow indicates the position of the full-length esterase. Incubation time intervals are indicated above corresponding lanes. Aptamers #3 and #23, directed against
RF1 from Thermus thermophilus, were the most efficiently binding aptamers against
E. coli RF1. Kd was approximatively 50 nM (see, Szkaradkiewicz (2002), FEBS Lett.
514, 90-95).
C: Influence of antibodies against RF1 from T. th. on C-terminal incorporation of CC-
Puromycin in the cell-free translation system programmed by pEst2. 2.5 μM [32P]p-
CC-puromycin (301 mCi/pmol) was used for in-vitro translation together with
Gel #2: anti-RF1 antiserum, dilution factor 1:30;
Gel #3: anti-RF1 antiserum, dilution factor 1 :300;
Gel #4: anti-RF1 antiserum, dilution factor 1 :3000.
The arrow indicates the position of the full-length esterase. Incubation time intervals are indicated above corresponding lanes.
Figure 9.
C-terminal incorporation of 32P-GC-Puromycin into in vitro produced esterase in the presence of aptamers and antibodies directed against RF1 and RF2. The cell-free translation system was programmed by pEst2 and 7 μM [32P]-GC-puromycin was used for in-vitro translation together with
1. no aptamer or antibody added;
2. 40 nM apamer #3 added as an RNA sequence
3. 40 nM apamer #23 added as an RNA sequence
4. Antibodies against T. th. RF1 (2μl of antiserum per 30 μl of translation mixture).
5. Antibodies against E. coli RF1 (2μl of antiserum per 30 μl of translation mixture).
6. Antibodies against T. th. RF2 (2μl of antiserum per 30 μl of translation mixture).
7. Antibodies against E. coli RF2 (2μl of antiserum per 30 μl of translation mixture).
8. Aptamer #3 produced within reaction mixture by T7 transcription from DNA template. 9. Aptamer #23 produced within reaction mixture by T7 transcription from DNA template.
A. Radioactive image of the SDS-gel
B. Relative amount of [32P]-GC-puromycin incorporated into full-length esterase. Radioactivity in the bands of full-length esterase 2 was numeralized into arbitrary units by ImageQuaNT program.
Aptamers #3 and #23, directed aginst RF1 from Thermus thermophilus, were the most efficiently binding aptamers against E. coli RF1. Kd was approximatively 50 nM (see, Szkaradkiewicz (2002), FEBS Lett. 514, 90-95).
Figure 10.
Incorporation of labelled Pu-derivatives into esterase 2 and functional properties of the resulting esterase-Pu-derivative conjugates.
A: C-terminal incorporation of [32P]pGpCpPuromycin, Biotin-[32P]pCpPuromycin and
Cy3-[32P]pCpPuromycin into polypeptides during in vitro transcription/translation of plVEX 2.3d-Est2 in the presence of antibodies against RF1 from Thermus thermophilus.
Radioactive image of SDS-PAGE of 2 μL samples withdrawn from translation mixture after 90 min. of incubation in the presence of 6 μM Puromycin-derivative. Lane 1 , no puromycin derivative was added; lane 2, [32P]pGpCpPuromycin was added; lane 3,
Biotin-[32P]pCpPuromycin was added; lane 4, Cy3-[32P]pCpPuromycin was added.
Position of the full-length esterase is indicated by an arrow.
A': Enzymatic activity of the synthesized esterase. The samples were as described in
A.
B: Fluorescent assay of Esterase-Puromycin conjugates. In vitro translation reaction were performed: lane 1 , in the presence of the Cy3-CpPuromycin; lane 2, in the presence of GpCpPuromycin, lane 3, in the absence of puromycin derivative.
C: Coomassie-stained SDS gel of the samples described in B.
Figure 11.
Incorporation of puromycin, carrying on the 5'-position 32P-labelled oligonucleotides of different length, into C-terminus of esterase2. Radioactive image of the SDS-gel, lane 1 : control (without puromycin derivative), lane 2, 3, 4, 5, 6 are the images of reaction mixtures after incorporation of 2, 4, 7, 9 and 11 nucleotide long deoxyribonucleotides attached to the puromycin (for examples see Fig. 4 and Fig 23), respectively.
The C-terminal incorporation of different length oligonucleotides attached to puromycin into polypeptides during in vitro transcription/translation of pEst2 was performed in the presence of 2 μl anti RF1 antiserum per 30 μl of the reaction mixture. The oligonucleotides and their conjugates with esterase were 5'- phosphorylated after translation as described in experimental section. Radioactive image of SDS-PAGE of 1 μl samples withdrawn from translation mixture after 120 min. of incubation in the presence of different concentration of the puromycin derivative. The experiment was carried out in the presence of: lane 1 : no puromycin derivative; lane 2-6: translation mixture contained the puromycin derivatives as shown in Fig. 23 1b, 1c, 1d, 1e, 1f, respectively. Position of the full-length esterase is indicated by an arrow.
A) Translation assay contained 11 μM puromycin-modified deoxyoligonucleotides. In lane 2, 0.5 μl of translation mixture were loaded in order to decrease the signal from truncated products.
B) Translation assay contained 31 μM puromycin-modified deoxyoligonucleotides.
C) Translation assay contained 94 μM puromycin-modified deoxyoligonucleotides.
Figure 12.
Immobilisation of esterase conjugated with biotinylated Puromycin.
A: Enzymatic activities of the esterase2 conjugated with GpCpPuromycin (control) and of esterase2 having incorporated GpCpPuromycin carrying a biotin on the N4 of the 5'-cytosin residue and being immobilized to streptavidin-coated polystyrene plates.
B: Incorporation of CpCpPuromycin carrying a biotin on the N4 of the 5'-cytosin residue in cell-free translation system. The synthesized protein-DNA fusion was then be immobilized onto a streptavidin-coated polystrene surface. The control is the is an esterase without biotin on the Puromycin-pCpC end.
Figure 13. Immobilization of Esterase-Puromycin conjugates produced on streptavidin coated glass surface. Translation reaction was performed as described in Experimental section. Plate 1 is a control onto which 1μL purified esterase, prepared by purification from E. coli cells overexpressing the enzyme according to (Arkov (2002), J Bacteriol. 184, 5052-5057). On the plates 2, 3, and 4 one μL translation mixture containing no puromycin derivative (2), Biotin-CpPuromycin (3) and Biotin-CpPuromycin (4) together with anti-RF1 antibodies, respectively. After 90 min. at 37°C the plates were rinsed by tap-water and the esterase activity was determined by 2-naphthyl acetate assay and developed with Fast Blue BB salt.
Figure 14.
Plasmid map of pEst2_amb155 with internal amber and terminal opal stop codon.
Figure 15.
Nucleotide sequence of pEst2_amb155 (SEQ ID NO: 41). Bolded letters are the coding sequence of esterase 2 Ser155amber mutant.
Figure 16.
Substitution of the sense codon for catalytically important Serine 155 (AGC) into nonsense codon (UAG) in the mRNA for the esterase 2 from Alicyclobacillus acidocaldarius
A: Scheme of mutated mRNA
B: The esterase catalytic triad (Ser-His-Asp) structural organization (De Simone
(2000), J MoI. Biol 303, 761-771).
Figure 17.
In vitro synthesis of the pEst2_Amb155.
A: Kinetics of in vitro transcription/translation of the pEst2_Amb155; filled triangles: pEst2 (control) as a template; filled squares: pEst2_Amb155 as a template. The concentration of the newly synthesized proteins was determined by measurement of the TCA precipitatable [14C]leucine radioactivity in aliquots withdrawn at indicated time intervals. B: Radioactive image of the SDS-PAGE of 2 μl samples from the in vitro translation mixture after 120 min. incubation time. Lane 1, pEst2_Amb155 as a template; lane 2, pEst2 (control) as a template.
C: Esterase activity of the in vitro synthesized polypeptides determined by hydrolysis of p-nitrophenyl acetate. Bar 1 , pEst2_Amb155 as a template; bar 2, pEst2 (control) as a template.
Figure 18.
Suppression of amber UAG stop codon by tRNASer sup in the coding region of esterase in the presence of anti-RF1 antibodies. Serine 155 is a residue assential for catalysis.
A: Autoradiography of 14C-labelled in vitro translation products by SDS PAGE of translated products: lines 1 , 2 and 3 amber UAG 155 as a template, line 4 is a control with Ser 155 codon as an template. Line 1 ; complete translation mixture (polypetide terminated at position 155), line 2; RF1 depleted by RF1 antibodies (nearly cognate aminoacyl-tRNAs suppress inefficiently the amber code, since essential serine is missing, no activity ), line 3; RF1 depleted by RF1 antibodies and amber suppressor tRNASer added (efficient synthesis of active, serine -155, esterase). B: Ezymatic activity of translated products measured by 4-nitrophenol assay. Activity bars correspond to SDS PAGE lanes.
Figure 19.
Suppression of amber stop codon by suppressor tRNASer(CUA) in the presence of antibodies against RF1 from Thermus thermophilus. Samples from the in vitro translation system programmed by pEst2_Amb155 were withdrawn after 160 minutes of incubation in the presence of [14C]leucine.
A: Translation was carried out in the absence of anti-RF1 antibodoes. Radioactive image of the gel after SDS-PAGE separation of 2 μl samples from the in vitro translation mixture is presented. Concentration of added suppressor tRNASer(CUA) was as follows: lane 1 , no tRNASer added, lane 2, 24 nM, lane 3, 120 nM, lane 4, 600 nM, lane 5, 2.5 μM, lane 6, 10 μM, lane 7, 25 μM tRNASer added. B: Translation was carried out in the presence of anti-RF1 antibodies. Radioactive image of the gel after SDS-PAGE separation of 2 μl samples from the in vitro translation mixture is presented. Concentration of added suppressor tRNASer was the same as described in (A).
C: Enzymatic activity of the in vitro produced esterase determined by hydrolysis of p- nitrophenyl acetate. Samples of 1 μl were used for detection. Grey bars, translation was carried out in the absence of anti-RF1 antibodies, black bars, translation was carried out in the presence of anti-RF1 antibodies. Concentration of added suppressor tRNASer(CUA) was performed as described in (A).
Figure 20.
In vitro protein selection (SPOT) from a library of mutated DNA matrices.
Figure 21.
C-terminal incorporation of [32P]pGpCpPuromycin into polypeptides during in vitro transcription/translation of pEst2 in the presence of antibodies raised against T. thermophilus RF1 and upon addition of purified E. coli RF1.
A) Radioactive image of SDS-PAGE of 2 μl samples withdrawn from translation mixture after 120 min of incubation in the presence of 7 μM [32P]GpCpPuromycin and different amounts of antibodies against RF1. Amount of added anti RF1 antiserum per 30 μl of the reaction mixture is indicated above corresponding lane. Position of the full-length esterase is indicated by arrow.
B) The dependence of the reaction yield on added amount of anti RF1 antibodies. The reaction yield was determined as X/Y*100 where X are pmols of
[32P]GpCpPuromycin incorporated into the full-length esterase (determined by Phosphoimager software); Y are pmols of active full-length synthesized esterase.
C) Radioactive image of SDS-PAGE of 2 μl samples withdrawn from translation mixture after 120 min of incubation in the presence of 7 μM [32P]GpCpPuromycin, 4μl of anti RF1 antiserum and different amounts of purified RF1. Concentration of added RF1 is indicated above corresponding lane. Position of the full-length esterase is indicated by arrow. D) The dependence of the reaction yield on added amount of RF1. The reaction yield was calculated as described in (B). The yield corresponding to value obtained in the absence of added E. coli RF1 is indicated left of the ordinate.
Figure 22.
A: Activity of the esterase conjugated with GpC(Biotin)pPuromycin immobilized to Streptavidin-Sepharose. In vitro transcription/translation was carried out as described in experimental section (Example 20) in the presence of: lane 1 : no antibodies, no puromycin derivative; lane 2: no antibodies, 7 μM of C(Biotin)pPuromycin; lane 3:2 μl of anti RF1 serum per 30 μl of reaction mixture, no puromycin derivative; lane 4: 2 μl of anti RF1 serum per 30 μl of reaction mixture, 7 μM of C(linker-NH2)pPuromycin; lane 5: 2 μl of anti RF1 serum per 30 μl of reaction mixture, 7 μM of C(Biotin)pPuromycin;
B: Activity of the esterase conjugated with C(Biotin)pPuromycin immobilized to streptavidin-coated polystyrene plates. The samples were as described in (C). Number 6 indicates untreated and empty well.
Figure 23.
Examples of derivatives of CpPuromycin modified on the 5^-end of the cytidine residue. The shown derivatives of CpPuromycin can be in the shown radioactive labelled form or in the corresponding non-radioactive labelled form.
The Examples illustrate the invention.
Example 1: Cloning of the release factor 1 of Thermus thermophilus (T.th.RFl)
Degenerated primers were used to amplify a prfA specific probe from Thermus thermophilus genomic DNA by PCR. Preparation of T. thermophilus genomic DNA and subsequent genomic PCR followed conventional protocols for mesophilic bacteria (Sambrook (2001), Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, NY, Vol. 1, 2, 3). A 50 mg bacterial pellet in an Eppendorf tube was resuspended in 565 μl TE buffer, 30 μl 10% SDS and 5 μl 20 mg/ml Proteinase K was added and incubated for 1 h at 37° C. Lysis was performed after addition of 100 μl 5 M NaCI and repeated uptaking and emptying the bacteria with a needle-equipped syringe by shearing forces. After addition of 80 μl of 10% Hexadecyltrimethylammoniumbromid (CTAB) in 0.7 M NaCI, 10 min incubation at 65° C and extraction with 700 μl Chloroform/lsoamylalkohol and 5 times with 700 μl Phenol/Chloroform/lsoamylalcolhol 25:24:1, genomic DNA was precipitated with Isoamylalcohol. 1μg DNA from the genomic DNA preparation was used in a 100 μl PCR reaction containing 5 μl of each 10 μM primer, 10 μl 15 mM MgCI__2 , 10 μl 10x Taq Pol buffer (10OmM Tris-HCI pH 8.8, 500 mM KCI, 15 mM MgCI2), 1 μl 1 U/μl Taq Polymerase, and cycled 30 times with 30 s at 95°, 30 s at 60° and 60 s at 72° after an initial 5 min denaturation at 95°.
With the amplified fragment, a 3.0 kbp fragment could be identified carrying the complete prfA sequence. The fragment was cloned into the plasmid pBluescript KS+ and the resulting plasmid pBlueK4b was sequenced. The sequence identified is identical to that of the literature (Ito (1997), Biochimie. 79, 287-292) and is shown in SEQ ID NO: 1. The corresponding amino acid sequence is shown in SEQ ID NO: 2.
Example 2: Overexpression of the release factor 1 of Thermus thermophilus in E. coli and purification of the same
The T.th.RF1 protein was heterologous overexpressed in E.coli and purified to homogeneity as judged by SDS PAGE and Maldi mass spectroscopy. In brief, after cell lysis of RF1 -overproducing E.coli cells by Lysozyme and/or Parr bomb treatment, the S 100 supernatant from the ultracentrifugation was concentrated by AMS- precipitation, dialyzed against 50 mM Tris/HCI pH 7.5, 10 mM MgCI2, 1 mM β- mercaptoethanol, 5% glycerol and used for Q-Sepharose FF (Amersham-Pharmacia) ionexchange chromatography running a gradient from 0 to 500 mM NaCI. RF1- containing fractions were pooled, 15 min at 65°C heat-treated removing most E.coli proteins - including heterologous E.coli RF1 - AMS-precipitated, dialyzed against 10 mM K-Phosphate buffer pH 6.8 and used for hydroxyapatite chromatography (Merck) running a gradient from 10 to 500 mM K-Phosphate pH 6.8. Pooled RF1 -fractions were ammonium sulfate-precipitated, dialyzed against 100 mM sodium acetate pH 5.75 and used for a final ionexchange chromatography on EMD-SO3 " -Tentakel (Merck) running a gradient from 0 to 2 M KCI. Recovered Thermus thermophilus RF1 was ammonium sulfate-precipitated, dialyzed against 100 mM Tris/HCI pH 7.5, 100 mM KCI, 5% glycerol, and stored at -20°C after adding an equal volume pure Glycerol.
Example 3: Preparation of polyclonal antibodies against the release factor 1 of Thermus thermophilus
Heterologous, in E.coli overexpressed and purified Thermus thermophilus RF1 was used to immunize two rabbits following the standard one month immunization- protocol at Eurogentec (Seraing, Belgium): A first immunization used the glycerinated protein mixed with incomplete Freund's adjuvant and a intradermic multisite injection at the rabbits back at day 0. Three boost immunizations at day 14, 30 and 60 followed with a small bleeding after 45 days and termination of the rabbits and final bleeding after 70 days. Vacutainer tubes were used to process blood samples and remove agglutinated blood clots.
After three boosts, serum was collected, centrifuged and the polyclonal antibodies were stored at -20 °C.
Example 4: Cloning of the release factor 1 from E. coli (E.C.RF1) and purification of the same
Cloning of E. coli RF1 was first described in Caskey ((1984), J. Bacterid., 158(1):
365-368) and in Weiss ((1984), 158(1): 362-364). The entire prfA operon was analyzed by Li five years later ((1989), Gene 82 (2):209-217).
The clones of E. coli RF1 used herein were a gift from the lab of Mans Ehrenberg
(Uppsala, Sweden) and were obtained as inserts in pET11a plasmids as described by Dincbas-Renqvist ((2000), EMBO J., 19, 6900-6907). The nucleotide sequences and the corresponding amino acid sequences of E. coli RF1 are shown in SEQ ID
NO: 5 and SEQ ID NO: 6, respectively.
Wild type E.coli RF1 cloned in pET11a was used to subclone the protein into pET28, adding a N-terminal (His)6 -tag. Overexpression in E.coli gave soluble RF1-(His)6 used to purify the protein. In brief, after harvested cells were resupended in 50 mM Na- phosphate pH 7.0, 300 mM NaCI, 10% glycerol, 10 mM imidazol and lysed with a microfluidizer, the S30 supernatant was loaded on Ni-NTA agarose (Qiagen, Hilgen, Germany) for affinity chromatography using 20 mM imidazol pH 8.0 for washing and 250 mM imidazol for elution of bound RF1. Following ammonium sulfate precipitation and dialysis against 50 mM Tris/HCI pH 7.5, 10 mM MgCI , 1 mM β-mercaptoethanol,
5% glycerol the protein solution was submitted to Q-Sepharose FF (Amersham- Pharmacia, Freiburg, Germany) ion exchange chromatography running a linear gradient from 0 to 600 mM KCI. RF1-containing fractions were pooled, precipitated with ammonium sulfate, dialyzed against 10 mM K-phosphate pH 6.8 and used for hydroxyapatite chromatography (Merck, Darmstadt, Germany) running a linear gradient from 10 to 200 mM K-phosphate pH 6.8. Pooled RF1 -fractions were precipitated by ammonium sulfate, dialyzed against 100 mM Tris/HCI pH 7.5, 200 mM KCI, 20 mM MgCI2, 1 mM β-mercaptoethanol, 5% glycerol, and stored at -20°C after adding an equal volume pure glycerol.
Example 5: Preparation of polyclonal antibodies against the release factor 1 of E. coli
Antibodies against E. coli RF1 were processed as in Example 3 using the antibody service of Genaxxon Bioscience (Stafflangen, Germany). However, a one-month protocol with immunizations at day 0, 14 and 30 as well as a bleeding at day 45 was followed.
Example 6: Preparation of the plasmid plVEX 2.3d-Est2 (pEst2) comprising a cDNA encoding the esterase 2 from Alicyclobacillus acidocaldarius (Est2)
Plasmid pT7SCII containing esterase 2 (Est2) was provided by G. Manco, Nappies,
Italy (Manco (1998), Biochem. J, 332 (Pt 1), 203-212). The est2 gene was amplified by using pT7SCII-esterase as template, recombinant Tag-polymerase, and two synthetic oligonucleotides, estfor (5'- CCATGGCGCTCGATCCCGTCATTCAGC -3'; SEQ ID NO: 42) and estrev (5'- GAGCTCCTAGGCCAGCGCGTCTCG -3'; SEQ ID NO: 43) in a 30-cycle polymerase chain reaction (1 min at 95°C, 30 sec 60°C, and 1 min at
72°C).
The primer estfor was designed to introduce a Λ/col restriction site (underlined) at the initiation site which also leads to a C to G exchange (bold) in the coding sequence
(proline at position 2 changes to alanine). This amino acid replacement has no effect on structure or function of the enzyme. Primer estrev introduces a Sac\ restriction site (underlined) downstream from the UAG stop codon (bold). The PCR product was eluted from an agarose gel and ligated into pGEM-T vector (Promega) and completely sequenced to verify that only desired mutations were introduced. The obtained plasmid was then digested with Λ/col and Sacl, the cloned fragment was eluted from an agarose gel and ligated into Λ/col-Sacl-linearized in vitro-translation- vector plVEX 2.3d (Roche Diagnostics, Mannheim, Germany). The resulting plasmid, plVEX 2.3d-Est2 (pEst2), was used for in vitro translation. A map of pEst2 is shown in Fig. 2, the corresponding nucleotide sequence is shown in Fig. 3 (SEQ ID NO: 40.
Example 7: Purification of the plasmid plVEX 2.3d-Est2 (pEst2)
The plasmids for coupled in vitro transcription/translation were purified with modified PEG method (Nicoletti (1993), Biotechniques, 14, 532-4, 536). Therefor, a 12.5 ml cell culture, containing the plasmid was harvested and resuspended in 240 μL of 25 mM Tris/HCI pH 8.0, 50 mM glucose, 10 mM EDTA. Then 600 μL of 0.2 N NaOH, 1 % (w/v) SDS was added. The tube was gently turned over for several times and incubated for 4 minutes at room temperature. 450 μL of 3.6 M NaOAc, pH 5.0 was added and the suspension was gently mixed by turning over the tube for 20 times and incubated for 4 minutes at room temperature. Cell debris was removed by centrifugation at 16,000 g for 10 minutes and the supernatant was mixed with 400 μL of 40 % PEG 6000 and kept on ice for one hour. The sample was centrifuged at 16,600 g for 10 minutes and the pellet was completely dissolved in 150 μL ddH2O. After addition of 300 μL of saturated NH4Ac the suspension was incubated for 15 minutes on ice and then centrifuged at 16,600 g for 10 minutes at room temperature. The supernatant was mixed with 300 μL of isopropanol and incubated for 15 minutes at room temperature. After centrifugation at 16,600 g for 10 min, the DNA pellet was washed two times with 75% ethanol, dried and dissolved in distilled water.
Example 8: Preparation and radioactive labelling of the puromycine-derivatives of the present invention
All puromycin derivatives were synthesized by standard phosphoramidite chemistry (Berg, Tymoczko, Stryer, Biochemistry, 5th Edition, Freeman Co. New York, 2001 pp. 148-149) by Purimex, Staufenberg, Germany. The synthesis of the puromycin dinucleotide 5'-dC(N4-TEG-NH2)pPuromycin-3' was accomplished by coupling of N4 alkylamino synthon (dC(N4-TEG-NH-TFA)-phosphoramidit, provided by ChemGenes, Wilmington, MA01887 U.S.A., Cat-No. CLP-1329, Formula:
Figure imgf000068_0001
with 5'-Dimethoxytrityl-N-trifluroacetyl-puromycin^'-succinyl-lcaadong chain alkylamino)-CPG (provided by GlenResearch, Sterling, VA 20164 U.S.A., Cat.No. 20- 4040-xx, Formula:
Figure imgf000069_0001
The resulting 5'-dimethoxytrityl-protected dinucleotide was cleaved from the CPG matrix by 32% ammonium hydroxide and left under this condition at 65°C for 1h in order to achieve total deprotection. Subsequently the dinucleotide was purified by HPLC and the trityl group was removed by treatment with 80% aqueous acetic acid solution. The final purrification was achieved by two HPLC steps. The sample was concentrated by evaporation and desalted by passing through a SepPac cartridge. Concentration of the puromycin derivatives in the in vitro translation assay was 7μM, unless otherwise indicated. Examples for the puromycin derivatives that were used within the present application are shown in Fig. 4, Fig. 5 and 23.
To monitor the incorporation of puromycin-derivatives during translation reactions, the used puromycin derivatives were labelled with [γ-32P]ATP at the 5'-end by 32P in the following way.
The reaction mixture (10 μl) contains 0.8 U/μl T4-polynucleotide kinase, 4 μM ATP, 0.2 μM [γ-32P]ATP (10 μCi, 4950 mCi/mmol, Hartmann Analytic, Braunschweig, Germany), 4 μM puromycin-modified oligonucleotide (Purimex, Staufenberg, Germany) in T4-polynucleotide kinase buffer (70 mM Tris/HCI (pH 7.6), 10 mM MgCI2, 5 mM DTT). Phosphorylation was carried out for 30 minutes at 37°C. Then the entire volume of the reaction mixture was mixed with 6.6 μl of up to 100 μM puromycin derivative. The resulting solution (up to 50 μM, 301 mCi/pmol) was used for in vitro translation experiments. When labeling was performed after translation, 1 μl translation mixture was used instead of 4 μM puromycin modified oligonucleotide.
Example 9: The SDS-PAGE (polyacrylamid gel electrophoresis) of the present invention
Protein pattern of the reaction mixture was analysed by SDS-PAGE (Schagger
(1987), Anal. Biochem., 166, 368-379). Aliquots were mixed with the sample buffer, incubated 5 min. at 95 °C and loaded onto 10% polyacrylamide gel. After the run, the gels were fixed with 15% formaldehyde in 60% methanol and stained with
Coomassie Blue G-250.
For imaging radioactivity, the dried gels were exposed to an imaging plate for radioactivity analysis with the Phosphorlmager SI (Molecular Dynamics, Sunnyvale,
USA).
Activity staining of the esterase in polyacrylamide gels after electrophoretic separation was performed according to (Higerd (1973), J. Bacteriol., 114, 1184-1192) with Fast Blue BB Salt and β-naphthyl-acetate.
Example 10: The esterase activity assays of the present invention
Determination of esterase activity was performed as described in Manco ((1998), Biochem. J., 332, 203-212) with minor modifications. Aliquots of 1 μl were withdrawn from transcription/translation mixture and mixed with 1 ml of 50 mM phosphate buffer pH 7.5 containing 0.2 mM p-nitrophenyl acetate. The time course of the esterase- catalyzed hydrolysis of p-nitrophenyl acetate was monitored by the production of p- nitrophenoxide at 405 nm in 1 cm path-length cells with UV-Spectral photometer DU 640 (Beckman, Fullerton, USA) at 25°C. Initial rates were calculated by linear least- square analysis of time courses comprising less than 10% of the total substrate turnover.
Staining of esterase activity in polyacrylamide gels was performed as described herein above (Example 9).
Example 11 : Depletion of the release factor 1 from E. coli in an E. coli cell-free translation system by precipitating and/or crosslinking said release factor 1 from E. coli with polyclonal antibodies against the release factor 1 of Thermus thermophilus and thereby increasing the incorporation of puromycine and/or its derivatives at the C-terminal nonsense codon of the esterase 2 (Est2) from Alicyclobacillus acidocaldarius
The kits used for in vitro translation experiments within the present application were evaluation size transcription/translation kits from RiNA GmbH, (Berlin, Germany) and the reaction was performed according to the manual provided by the supplier. [14C]Leucine (25 mCi/mmol, Amersham) was added up to 0.5 mM. The template (control vector or plVEX 2.3d-Est2) was added up to 5 nM. The reaction was started by transferring the reaction tube to the thermo shaker at 37 °C with 500 rpm agitation. When the translation reaction was carried out in the presence of antibodies against RF1 from Thermus theromphilus, different amounts of serum were added per 30 μl of the reaction mixture. Radioactively labeled puromycin derivatives were added up to 6 μM. Amount of newly synthesized protein was determined from radioactivity of hot 10% trichloroacetic acid precipitate.
As examples, detailed protocols of the in vitro translations performed within the present application are listed below.
Protocol for a standard in vitro translation (RiNA GmbH Kits):
For preparation of a 30 μl reaction mixture the following components should be combined on ice (given in order of mixing):
1. 5.1 μl of 1 mM [14C]LeU (54 mCi/mmol, Amersham)
2. 1 μl 10 mM Leu (supplied with the Kit)
3. 0.5 μl of RNase free water (supplied with the Kit)
4. 2.4 μl of E-mix (red lid, supplied with the Kit)
5. 9 μl of T-mix without Leu (blue lid, supplied with the Kit)
6. 10.5 μl of S-mix (yellow lid, supplied with the Kit)
7. 1.5 μl of the 10O nM template plasmid
The reaction mixture should be incubated at 37°C for 2 hours with agitation (500 rpm). Aliquots can be withdrawn at any desired time intervals. The reaction can be performed without radioactivity (Leu should be substituted with the same amount of water and T-mix with Leu (supplied with the Kit) should be used instead of one without. Protocol for in vitro translation (RiNA GmbH Kits) in the presence of anti-RF1 (T. thermophilus) antibodies:
For preparation of a 30 μl reaction mixture the following components should be combined on ice (given in order of mixing):
1. 5.1 μl of 1mM [14C]LeU (54 mCi/mmol, Amersham)
2. 1 μl 10 mM Leu (supplied with the Kit)
3. 2.4 μl of E-mix (red lid, supplied with the Kit)
4. 9 μl of T-mix without Leu (blue lid, supplied with the Kit)
5. 10.5 μl of S-mix (yellow lid, supplied with the Kit)
6. 2 μl of rabbit anti-RF1 antiserum
7. 0.1 μl of the 1.9 μM template plasmid
The reaction mixture should be incubated at 37°C for 2 hours with agitation (500 rpm). Aliquots can be withdrawn at any desired time intervals.
Protocol for in vitro translation (RiNA GmbH Kits) in the presence of anti-RF1 (T. thermophilus) antibodies and Puromycin derivatives:
For preparation of a 30 μl reaction mixture the following components should be combined on ice (given in order of mixing):
1. 1.5 μl of the 100 nM template plasmid
2. 2.4 μl of E-mix (red lid, supplied with the Kit)
3. 9 μl of T-mix WITH Leu (blue lid, supplied with the Kit)
4. 10.5 μl of S-mix (yellow lid, supplied with the Kit)
5. 2 μl of rabbit anti-RF1 antiserum
6. 4 μl of 50 μM solution of puromycin derivative
The reaction mixture should be incubated at 37°C for 2 hours with agitation (500 rpm). Aliquots can be withdrawn at any desired time intervals.
For instance, the standard in vitro translation system of the present application (without RF depleating agents (e. g. Antibodies against RF1 from Thermus thermophilus) or nonsense codon suppressing agents (e. g. puromycin derivatives and/or suppressor tRNAs)) comprises the following ingredients: - 3OS cell-free extract from E. coli (enzyme- and und ribosomal fraction);
- MgCI2 9-12 mM;
- DTT 10 mM;
- Amino acids, 200 μM each (For labelling, each amino acid can be applied as a 14C amino acid with a concentration of 100 μM (e.g. 14C-leucine))
- Rifampicin 0,02 mg/ml reaction mixture,
- Bulk-tRNA 600μg/ml reaction mixture,
- ATP,CTP,GTP,UTP, 1mM each,
- Phosphoenolpyruvate 10 mM;
- Acetylphosphate 10 mM;
- Pyruvatekinase 8 μg/ml reaction mixture;
- Plasmid 2 pmol/ml reaction mixture;
- T7 Polymerase 500 Units/ml reaction mixture;
- HEPES pH 7,6, 5O mM;
- Potassium acetate70 mM;
- Ammonium chloride 30 mM;
- EDTA pH 8,0 , 0,1 mM;
- Sodium azide 0,02 %;
- Polyethyleneglycol 4000 2 %;
- Protease inhibitors: aprotinin 10 μg/ml reaction mixture, leupeptin 5 μg/ml reaction mixture, pepstatin 5 μg/ml reaction mixture; and
- Folic acid 50 μg/ml reaction mixture.
In order to improve C-terminal incorporation of puromycin, the E. coli release factor 1 (RF 1) responsible for termination at the UAA and UAG stop codons was inactivated in the in vitro translation system from E. coli by rabbit antibodies specific against Thermus thermophilus RF1. Template DNA that encoded mRNA for synthesis of the esterase 2 from Alicyclobacillus acidocaldarius (Manco (1998), Biochem. J, 332 (Pt 1), 203-212) and ended by UAG stop codon, was used. The C-terminal labeling of the esterase was monitored by incorporation of [32P]pGpCpPuromycin into full-length protein (Figure 6A). The activity of the synthesized esterase was also determined (Figure 6B). The yield of conjugation of puromycin derivative with the esterase was very low in the presence of endogenous E. coli RF1 (Figure 6A, lane 1). However, deactivation of RF1 with RF1 -specific antibodies leads to a strong, at least 50-fold, stimulation of [32P]pGpCpPuromycin incorporation into the C-terminus and provided a puromycin-conjugated esterase in, at least, 80% yield (Figure 6B). The antibody concentration range in which the puromycin-mediated termination is switched-on is very narrow (compare lanes 5 and 6 Fig.6A). The estimated concentration of RF1 at which an efficient incorporation can be achieved is less then 10 nM. Figure 6 shows that only at high RF1 antibody concentration (lane 6), [32P]pGpCpPuromycin starts to be incorporated efficiently into full-length, active esterase. Further increase of antibody concentration does not improve the C-terminal incorporation of the drug.
Similar to [32P]pGpCpPuromycin the incorporation of [32P]pCpCpPuromycin is shown herein. The corresponding experiment with polyclonal antibodies against Thermus thermophilus RF1 that stimulate the incorporation of said Puromycin derivative, extended on the 5'-OH group by two cytidylic acid residues, into C-terminal position of the esterase is demonstrated in Fig. 7. An in vitro system derived from E. coli as listed above was used in this experiment. Similar result was achieved by antibodies against E. coli RF1. The yield of puromycin incorporation increased at least 50 fold in the presence of anti-RF1 serum from rabbits as compared to untreated commercially available translation systems (Fig. 7).
Further, the efficiency of the incorporation of puromycin-derivatives into the C- terminus of esterase 2 in the presence of different dilutions of antiserum against Thermus thermophilus RF1 (Fig. 8C; Fig. 9, lane 4) was compared to the efficiency of the incorporation of puromycin-derivatives in the absence of RF depleting agents (Fig. 8A; Fig. 9, lane 1), antiserum against Thermus thermophilus RF2 as putative RF depleting agents (Fig. 9, lane 6), antiserum against E. coli RF1 and E. coli RF2 as putative RF depleting agents (Fig. 9, lane 5; Fig. 9, lane 7) and aptamers against E. coli RF1 (Szkaradkiewicz , (2002), FEBS Lett. 514, 90-95) as putative RF depleting agents (Fig. 8B; Fig. 9, lanes 2, 3, 8 and 9).
Following conclusions can be drawn from the accordant results. First, incorporation of puromycin analogue in the absence of antibodies against Thermus thermophilus RF1 is very poor (Fig. 8A; Fig. 9, lane 1). If at all it occurs preferentially at truncated products. Second, there is no stimulation of puromycin incorporation into full-length protein by RNA aptamers directed against RF1 (Fig. 8B; Fig. 9, lanes 2, 3, 8 and 9). Third, polyclonal antibodies against Thermus thermophilus RF1 are efficient in stimulation of Puromycin incorporation (Fig. 8C; Fig. 9, lane 4). The serum containing antibodies directed against E. coli RF1 is at 1 :30 dilution as effective for the depletion of E. coli RF1 as the serum containing anti Thermus thermophilus RF1 antibodies at 1 :3.000 dilution (Fig. 8C, #4).
The RF1 deficient in vitro translation system gives a possibility to be employed for incorporation of longer oligonucleotides via C-terminal puromycin reaction (Starck, 2002 loc. cit). However, it is also shown herein, that oligonucleotides being covalently bound to the 5"-end of puromycin that are longer than 5-6 nucleotide residues can not enter the ribosomal A-site in EF-Tu. GTP independent manner (Fig. 11).
Starting from a puromycin 5'-extended by one nucleotide (Fig. 23, 1b), the 5'-end was consecutively elongated (Fig. 23, 1c-1g) to test the effect of oligonucleotide length on the C-terminal incorporation (Fig. 11). A change in the sequence of puromycin 5'-extension leads to remarkable effects. pCpCpPuromycin (Fig. 23, 1b) gives rise to much stronger premature termination (Fig. 11A lane 2) as compared to pGpCpPuromycin (Fig. 23, 1a; (Fig. 10 A/A' lane 2). This effect can be explained by the homology of pCpCpPuromycin (Fig. 23, 1b) to the CpCpA end of the natural substrate aminoacyl-tRNA leading to efficient interaction with the A-site of peptidyl transferase (Chladek, Angew.Chem.lnt.Ed. 1985, 24 371-391.). Conjugation of puromycin with four nucleotides provides efficient C-terminal incorporation (Fig. 11 lane 3) that gradually decreases with the length of attached nucleotide (Fig. 11 lanes 4-6). Incorporation of a undecamer is hardly detectable. Increase of the puromycin derivative concentration does not improve the C-terminal incorporation in case of oligonucleotides longer than four (Fig. 11 B and C). These results correlate with the observation that the 5'-extension of puromycin by an oligonucleotide leads to an almost complete inability of the antibiotic to inhibit protein biosynthesis (Starck, 2002, loc. cit). Simple sterical limitations for this effect seem to be improbable since the ribosomal A-site can accommodate aminoacyl-tRNA that is much larger in size compared to the oligonucleotide-puromycin derivatives. Accommodation of aminoacyl-tRNA to the A-site is, however, a multistep process in which the details of aminoacyl-tRNA structure have important functions, which can not be mimicked by puromycin.
There is, however, a possibility to circumvent the problem that oligonucleotides being covalently bound to the 5*-end of puromycin and are longer than 5-6 nucleotide residues can not enter the ribosomal A-site. Inter alia this problem was solved by the present invention.
Instead of covalent attachment of RNA to the 5 '-position of puromycin an alternative strategy by which the RNA (mRNA) or other functional groups are attached directly or via a linker to the nucleobases of puromycine-derived oligonucleotides (e.g. CpCpPu or CpPu) was used. Example for this type of conjugation are demonstrated in Fig. 5. A direct synthesis of puromycin-DNA conjugates can be achieved by synthons that serve as linkers between Cp-Pu and any oligonucleotide synthesised by a standard DNA synthesis.
As an example for incorporation of puromycin derivatives, having covalently attached functional groups directly or via a linker to the nucleobases of puromycine-derived oligonucleotides, into proteins, the in vitro synthesis of esterase 2 from Alicyclobacillus acidocaldarius with C-terminal Biotin-CpPu is demonstrated (Fig 12B). The resulting protein was purified on a streptavidin affinity material under retention of full esterase activity. When CpPu, without biotin is used as an control in the in vitro translation mixture no significant retention of the esterase on streptavidine affinity matrix could be recorded (Fig 12B). Binding of the biotinylated esterase was performed to streptavidin coated polystyrene plates (Nunc GmbH, Wiesbaden, Germany) according to the manual provided by the supplier. Esterase activity was monitored in 5OmM phosphate buffer with 0.2 mM p-Nitrophenyl acetate by μQuant Universal Mircoplate Spectrometer (Bio-Tek Instruments, Vermont, USA).
Further, to explore the possibility to use puromycin in the absence of active RF1 as a vehicle to carry reporter groups, affinity tags or even large oligomeric structures to be linked with a polypeptide, biotin or Cy3 fluorophore were both covalently attached via the exocyclic N4 of the cytidine residue to 5'[32P]pCpPuromycin (Fig. 4; Fig. 5). These substrates were incorporated with the same efficiency into full-length polypeptide as pGpCpPuromycin (Fig. 10A). The C-terminal incorporation of puromycin derivatives did not lead to any detectable loss of activity of the esterase.
The attachment of Cy3 fluorophor to the esterase was visualized not only by radioactive labelling of the derivative (Figure 10A, lane 2) but also by monitoring the fluorescence directly after SDS-PAGE of total protein in polyacrylamide gel (Figure 10B). The fluorescent band in line 1 of Figure 10B coincides with the full-length esterase. The corresponding controls, when puromycin derivatives without fluorescent chromophore were incorporated, show, as expected, no fluorescent band (Figure 10B, lines 2 and 3).
Thus, these experiments demonstrate, that puromycin can be used as a delivery carrier of different chemical functional groups for specific C-terminal labelling of protein in high yield.
Example 12: Immobilization of esterase 2 having incorporated a biotinolated puromycine-derivative on streptavidin coated polystyrene surfaces
In vitro translation in the presence of biotinylated puromycin derivative and antibodies against RF1 can be used in situ for immobilization of a protein to streptavidine-coated solid surfaces without protein purification. This is demonstrated in Fig 12 and Fig. 13 with esterase 2 from Alicyclobacillus acidocaldarius as a reporter enzyme. For instance, the attachment of biotin to the esterase was further demonstrated by immobilization of the resulting protein-puromycin-biotin conjugate to the surface of streptavidin-coated polystyrene plates (Fig. 12A). The streptavidin coated plate treated with C-terminally biotinylated esterase (having incorporated Biotin- pGpCpPuromycin) was enzymatically active (Fig. 12A, 2nd bar) whereas the control plate that was treated by esterase conjugated with the GpCpPuromycin did not bind the enzyme and had considerably lower activity (Fig. 12A, 1st bar). The residual esterase activity in this control experiment was probably due to unspecific adsorption of the esterase to the polystyrene surface.
The binding of the biotinylated esterase to streptavidin coated polystyrene plates (Nunc GmbH, Wiesbaden, Germany) was performed according to the manual provided by the supplier. Esterase activity was monitored in 5OmM phosphate buffer with 0.2 mM p-Nitrophenyl acetate by μQuant Universal Mircoplate Spectrometer (Bio-Tek Instruments, Vermont, USA).
A detailed protocoll of the binding of biotinylated esterase to polystyrene plates is listed below:
• 1 μl from a translation mixture was mixed with 99 μl of 100 mM Tris-HCI (pH 7.5), 150 mM NaCI and 0,1% Tween 20
• The above prepared mixture was transferred to a polystyrene plate and sealed with parafilm
• The polystyrene plate was incubated for 30 minutes at 50°C
• Solution was removed, and the well was washed with 100 mM Tris-HCI (pH 7.5), 150 mM NaCI and 0,1% Tween 20 for 3 times. Then the well was soaked with the same buffer at room temperature for 5 minutes. Afterwards it was washed another 3 times and then emptied.
• 200 μl 50 mM NaPO4 (pH 7.5) and 2 mM para-nitrophenylacetate were added, and the absorbance at 405 nm was recorded.
Example 13: Immobilization of esterase 2 having incorporated a biotinylated puromycine-derivative on streptavidin coated glass surfaces
Further, the attachment of biotin to the esterase was demonstrated by immobilization of the resulting protein-puromycin-biotin conjugate to the surface of streptavidin- coated glass plates (Fig. 13). The attachment to said glass plates (Greiner Bio-one) was performed as described above for streptavidine-coated polystyrene plates (Example 12).
At position 1 of Fig. 13, 1μL esterase purified from overexpressing E. coli strain (Manco (2000), Arch. Biochem. Biophys., 373, 182-192) was applied to streptavidin- coated glass plates. At positions 2, 3 and 4 in vitro translation, programmed by esterase gene, was performed directly "on spot" in 1 μL translation mixture containing no puromycin derivative on the streptavidine-coated glass plates. The translation mixture without Biotin-CpPuromycin and RF1 antibody was placed on spot 2, translation mixture in the presence of Biotin-CpPuromycin on spot 3 and the translation mixture with both, Biotin-CpPuromycin and RF1 antibody on spot 4. After translation performed for 90 min. at 37°C in a cell free translation System as described herein above (Ex. 11). The unbound components were removed by rinsing the plates with tap-water. The residual activity of biotinylated esterase bound to the streptavidine coated glass plates (Greiner Bio-One, Frickenhausen, Germany) was determined by applying 2 μL solution composed of 20 mg of 2-naphthyl acetate dissolved in 1 ml_ of acetone and 150 mg of Fast Blue BB salt suspended in 4 mL 100 mM Tris/HCI pH 7.5, to the surface of the plate. Esterase containing spots became brown-coloured after few minutes. The reaction was stopped by rinsing the gel in tap water.
As demonstrated in Fig. 13, the control experiments on spots 1 and 2 show no esterase activity. Only very little esterase activity could be detected on spot 3 where the translation was performed in the presence of biotin-puromycin and in the absence of antibodies against RF1. Obviously, the competition of the puromycin-derivative with RF1 for the AUG triplet-coded A-site prevented an effective incorporation. Only after inactivation of RF1 with antibodies (Fig 13, plate 4) significant amount of esterase has been immobilized.
Example 14: Preparation of the plasmid pEst2_amb155 comprising a cDNA encoding an AGC155→TAG155-mutated esterase 2 (EST2) from Alicyclobacillus acidocaldarius
The ACG triplet in the est2 mRNA of Alicyclobacillus acidocaldarius esterase coding for serine-155 was replaced by the RF1 stop codon UAG (amber), while the stop codon at the end of the mRNA was substituted for UGA (opal) codon that promotes RF2-dependent termination (Fig. 16A).
Therefore, site-directed mutagenesis was performed on the esterase 2 gene (Est2) in plVEX 2.3d-Est2 (pEst2) plasmid by the overlap extension method. Two separate PCR reactions was carried out using (1) T7 promotor primer (5'- TAATACGACTCACTATAGGG-3'; SEQ ID NO: 44) and EstS115x_rev (5'- ATTCCCTCCGGCCTAGTCTCCGCCGACCGCGATGC-3'; SEQ ID NO: 45); (2) EstS115x_for (5'-CGGTCGGCGGAGACTAGGCCGGAGGGAATCTTGCC-3'; SEQ ID NO: 46) and T7 terminator primer (5'- CTAGTTATTGCTCAGCGGTG-3'; SEQ ID NO: 47). The mutated codons are bolded and the serine codon at position 155 of amino acid sequence of the esterase was changed to RF1 stop codon (TAG) mutation. The PCR fragments were fused by another PCR using T7 promotor and T7 terminator primers. The fused PCR product was digested with NcollSacl and ligated into Ncol/Sacl digested plVEX 2.3d vector. The ligation mixture was transformed into E.coli strain XL-1 Blue. The plasmid DNA was isolated from clones and sequenced before use. The resulting plasmid pEst2_amb155 was used for in vitro translation. A map of pEst2_amb155 is shown in Fig. 14, the corresponding nucleotide sequence is shown in Fig. 15 (SEQ ID NO: 41). The plasmid was purified as described herein above (Ex. 7)
Example 15: Preparation of suppressor tRNASerCUA
Within the present application, the used suppressor tRNASerCUA was prepared as follows.
The gene of tRNASerCUA was constructed by PCR using primers tSer-amber1 (5'- GGAATTCTAATACGACTCACTATAGGAGAGATGCC-3'; SEQ ID NO: 48), tSer- amber2 (5'-GTCCGTTCAGCCGCTCCGGCATCTCTCCTATAGTG-3'; SEQ ID NO: 49), tSer-amber3 (5'-CTCCGGTTTTAGAGACCGGTCCGTTCAGCCGCTCC-3'; SEQ ID NO: 50), tSer-amber4 (5'-CCGGTA GAGTTGCCCCTACTCCGGTTTTA GA GA CC- 3'; SEQ ID NO: 51), tSer-amber5 (5'-
GAGAGGGGGATTTGAACCCCCGGTAGAGTTGCCCC-3'; SEQ ID NO: 52), tSer- amberβ (5'-AAGCTTGGATGGATCACCTGGCGGAGAGAGGGGGATTTGAAC-3'; SEQ ID NO: 53). Bolded letters are T7 promotor and italic letters are the gene of tRNASerCUA. The mutated anticodon is underlined. The conditions of the performed PCR were 95°C denaturation for 30 seconds, 50°C annealing for 30 seconds and 72°C polymerization for 30 seconds; 25 cycles were performed. The primer concentration was about 1 nM. DNTP concentration was 0.4 mM. The sequence of suppressor tRNA is based on a tRNASer from E.coli (tRNA databank number DS 1660) with a CUA mutation from position 34 to 36. The PCR product was cloned in a pGEM-T vector. The resulting plasmid ptSer-amber was sequenced and used as a template for the following PCR. The PCR was performed with primers tSer-amber1 and M13_rev (5'-CAGGAAACAGCTATGACC-3'; SEQ ID NO: 54). The PCR product was digested with BstNI for a CCA end and used as the template for in vitro transcription. The transcripts were purified by urea polyacrylamide gel electrophoresis and stored at -20°C. Example 16: Depletion of the release factor 1 from E. coli in an E. coli cell-free translation system by precipitating and/or crosslinking said release factor 1 from E. coli with polyclonal antibodies against the release factor 1 of Thermus thermophilus and thereby increasing the incorporation of an (unnatural) amino acid delivered by aminoacyl suppressor tRNACUA at an internal nonsense codon of an AGC155→TAG155-mutated esterase 2 (EST2) from Alicyclobacillus acidocaldarius
The kits used for in vitro translation experiments within the present application were evaluation size transcription/translation kits from RiNA GmbH (Berlin, Germany) and the reaction was performed according to the manual provided by the supplier. [14C]L- Leucine (54 mCi/mmol) was added up to 160 μM along with leucine resulting in 0.5 mM total concentration. The templates pEst2 and pEst2_amb155 were added up to 5 nM concentrations. The reaction was performed at 37 °C with agitation. Aliquots, 3μL, were withdrawn at different time intervals and the newly synthesized protein was determined by radioactivity measurement in 10% trichloroacetic acid precipitate. Protein composition was analysed by SDS-PAG]. The gels were fixed with 15% formaldehyde in 60% methanol and stained with Coomassie Blue G-250. The dried gels were exposed to an imaging plate for radioactivity analysis with the Phosphorlmager SI (Molecular Dynamics, Sunnyvale, USA).
The in vitro translations were performed according to the protocols shown in example 11 with minor modifications. As an example a detailed protocol of the in vitro translation performed in the presence of anti-RF1 (T. thermophilus) antibodies and suppressor tRNA is listed below.
Protocol for in vitro translation (RiNA GmbH Kits) in the presence of anti-RF1 (T. thermophilus) antibodies and suppressor tRNA:
For preparation of a 30 μl reaction the following components should be combined on ice (given in order of mixing):
1. 5.1 μl of 926 μM [14C]LeU (54 mCi/mmol, Amersham)
2. 1 μl 10 mM Leu (supplied with the Kit)
3. 2.4 μl of E-mix (red lid, supplied with the Kit)
4. 9 μl of T-mix without Leu (blue lid, supplied with the Kit) 5. 10.5 μl of S-mix (yellow lid, supplied with the Kit)
6. 2 μl of rabbit anti-RF1 antiserum
7. 0.5 μl of 1.52 μM tRNASer(CUA) (can be varied in 20 fold range)
8. 0.1 μl of the 1.9 μM template plasmid
The reaction mixture should be incubated at 37°C for 2 hours with agitation (500 rpm). Aliquots can be withdrawn at any desired time intervals.
The standard in vitro translation system (without RF depleating agents (e. g. Antibodies against RF1 from Thermus thermophilus) or nonsense codon suppressing agents (e. g. puromycin derivatives and/or suppressor tRNAs) of the protocol listed above comprises the following ingredients:
- 3OS cell-free extract from E. coli (enzyme- and und ribosomal fraction);
- MgCI2 9-12 mM;
- DTT 10 mM;
- Amino acids, 200 μM each (For labelling, each amino acid can be applied as a 14C amino acid with a concentration of 100 μM (e.g. 14C-leucine))
- Rifampicin 0,02 mg/ml reaction mixture,
- Bulk-tRNA 600μg/ml reaction mixture,
- ATP,CTP,GTP,UTP, 1mM each,
- Phosphoenolpyruvate 10 mM;
- Acetylphosphate 10 mM;
- Pyruvatekinase 8 μg/ml reaction mixture;
- Plasmid 2 pmol/ml reaction mixture;
- T7 Polymerase 500 Units/ml reaction mixture;
- HEPES pH 7,6, 5O mM;
- Potassium acetate70 mM;
- Ammonium chloride 30 mM;
- EDTA pH 8,0 , 0,1 mM;
- Sodium azide 0,02 %;
- Polyethyleneglycol 4000 2 %;
- Protease inhibitors: aprotinin 10 μg/ml reaction mixture, leupeptin 5 μg/ml reaction mixture, pepstatin 5 μg/ml reaction mixture; and - Folic acid 50 μg/ml reaction mixture.
Using the construct of Example 14 as a template for in vitro protein synthesis, the suppression of the amber codon was studied by SDS-PAGE of the full-length esterase production (Fig. 17B) and by measurement of the catalytic activity of the in vitro synthesized esterase (Fig. 17C). Translation of est2 mRNA(Ser-155) and est2 mRNA(amber-155) as measured by [14C]leucine incorporation into polypeptide chain provides a protein of 34.4 and 17.3 kDa (Fig. 17B), respectively, approximately with the same efficiency (Fig. 17A). The amber mutation in position 155 leads to complete termination and synthesis of 17.3 kDa protein void of esterase activity (Fig. 17B; lane 1 and Fig. 17C).
The experiment shown in Figure 18 also demonstrates the effect of polyclonal antibodies raised against RF1 in promoting a suppression of an AUG amber triplet during E. coli in vitro translation. In figure 16A the two DNA constructs, one with Ser 155 and the other with amber 155 are shown. SDS-PAGE and the autoradiography of the 14C-labelled products of in vitro synthesis are shown in Fig 18A. The esterase acticity of the in vitro synthetised products is demonstrated in Fig 18B. Esterase 2 from Alicyclobacillus acidocaldarius has an essential serine residue in position 155 of its sequence, that is part of the catalytic triad (Ser-His-Asp).
The hydroxyl of this serine side chain is the acceptor of the acyl residue during the hydrolysis of the ester bond. The lane 4 of the SDS -PAGE experiment shown in Fig.18A demonstrates the synthesis of the active full-length protein when a DNA with an normal AGC coding for Ser155 is present. The replacement of this triplet by an amber stop codon UAG leads to a production of a inactive polypeptide with 154 amino acids (Fig 18A, lane 1 , Fig 18B, column 1). This is a result of the recognition of UAG triplet by endogenous RF1 and hydrolysis of peptidyl-tRNA. Addition of anti- RF1 antibodies suppress the termination at UAG (Fig 18A1 lane 2) and a small amount of full-length protein visible in lane 2 is produced. This is a result of a suppression of the UAG by endogenous aminoacyl-tRNAs that can, to some extent, read UAG. Since several different amino acids canbe incorporated to position 155 in this manner, the activity of the full-length polypeptide is low (Fig 18B, column 2). Finally, the addition of suppressor Ser-tRNA possessing the anticodon AUC promotes the synthesis of full-length active esterase provided the endogenous RF1 was removed by antibodies (Fig 18A, lane 3, Fig. 18B, column 3). The experiments in Fig 18. demonstrate for a first time an efficient, nearly complete suppression of UAG stop codons. This allows now to use suppressor tRNASer or other amber supressor tRNA as a vehicle to incorporate unnatural and modified amino acids into any protein in vitro. This possibility to extend the genetic code wit high efficiency has far-reaching consequences and opens the way for labelling of proteins and preparation of proteins with many new properties.
Addition of increasing amounts of amber suppressor tRNASer(CUA) to the translation mixture that is programmed by est2 mRNA(amber-155) gives rise to the synthesis of the full size polypeptide chain. The required concentration of tRNASer(CUA) for production of the full-length protein in maximal yield is about 5 μM (Fig 19A). This value is in the range of concentrations of tRNA isoacceptors in E. coli cells (Dong (1996), J. MoI. Biol. 260, 649-663). Although, the amber suppressor Ser-tRNASer(CUA), with an anticodon complementary to UAG and presented in complex with EF-Tu. GTP and has the optimal prerequisites to enter the UAG-programmed A-site, it has still to compete with endogenous RF1. As demonstrated in Fig. 19A and C this competition starts to be efficient only at μM concentration of Ser- tRNASer(CUA). The cellular concentration of RF1 is similar to that of tRNA isoacceptors (Dong (1996), J. MoI. Biol. 260, 649-663; Adamski (1994), J. MoI. Biol 238, 302-308). However, during preparations of cellular extracts for in vitro translation the concentrations of all cellular components drop as compared to the situation in cytoplasm. Whereas the tRNA concentration in the in vitro translation system was adjusted by addition of bulk tRNA to 50 μM, the concentration of RF1 becomes about 50 fold lower as compared with the situation in vivo. It follows, that the average final concentration of a single aminoacyl-tRNA isoacceptor and RF1 in the in vitro translation mixture is about 1μM and 20 nM, respectively. The need for high Ser- tRNASer(CUA) concentrations to compete for RF1 probably reflects the different affinity for the ribosomal A-site of these alternative substrates. At concentrations higher than 1 μM, however, Ser- tRNASer(CUA) already starts to compete also with near-cognate aminoacyl-tRNAs for codon-specified binding to the A-site and the serine becomes misincorporated into several other positions of the polypeptide chain. This leads to accumulation of errors and loss of protein functionality, i.e. inactive enzyme (Fig. 19C). At very high tRNASer(CUA) concentrations the yield of the 17.3 and 34 kDa polypeptides drops, probably due to frameshifting and premature termination, and the [14C]leucine radioactivity becomes distributed between polypeptides of different lengths (Fig. 19A, lines 6 and 7).
Completely different is the situation in the absence of RF1 that can be efficiently deactivated by addition of antibodies against Thermus thermophilus RF1 to the E. coli in vitro translation system. Absence of RF1 leads to stimulation of UAG suppression by near-cognate endogenous tRNAs (compare Fig. 19A, line 1 and Fig. 19B, line 1). Thus, in the absence of RF1 (Fig. 19B) the synthesis of 17 kDa polypeptide substantially decreases as compared to the translation in the complete system (Fig. 19A) and only a small amount of mostly inactive, full-length protein is synthesized.
As compared to the complete system (Fig. 19A), in the absence of RF1 the concentration of tRNASer(CUA) required to achieve full UAG suppression and synthesis of active full-length esterase 2 from est2 mRNA(amber-155) drops dramatically (Fig. 19B). Already at 24 nM tRNASer(CUA) in the translation mixture the synthesis of the full- length (34 kDa) polypeptide becomes efficient. The esterase synthesized under these conditions is fully active. The yield of the synthesized enzyme and its activity are identical to the esterase obtained by translation of the wild-type est2 mRNA (Ser- 155). The yield of active esterase remains high up to 1 μM concentration of tRNASer(CUA) (Fig. 19C1 black bars 2-5). Further increase of the suppressor tRNA concentration in the translation mixture results in drop of protein production along with loss of enzymatic activity. In the high tRNASer(CUA) concentration range there is a coincidence between the data presented in Fig. 19A and 19B.
Thus, it was demonstrated that in the absence of RF1 the suppressor Ser- tRNASer(CUA) is efficiently bound to the A-site of UAG-programmed ribosomes. This leads to complete suppression of UAG codon and to incorporation of the catalytically essential serine-155 into the enzyme. At high Ser- tRNASer(CUA)-EF-Tu.GTP concentrations, the competition with other aminoacyl-tRNAΕF-Tu.GTP ternary complexes leads to misreading of near-cognate codons and results in synthesis of error prone or incomplete polypeptide chains void of enzymatic activity. Thus, the use of est2 mRNA(amber 155) as a template and the possibility to deactivate the endogenous RF1 in the in vitro translation system by RF1 antibodies permits an optimal adjustment of RF1 and tRNASer(CUA) concentrations to achieve a complete suppression and at the same time a maximal retention of enzymatic activity of the esterase.
Example 17: Preparation of protein arrays by using the ,,on spot" in vitro protein biosynthesis technology of the present invention
The DNA, that contains
• T7 RNA polymerase promoter,
• translation enhancer sequence,
• ribosomal binding sequence,
• an initiation triplet code,
• the sequence coding for the particular protein
• and a termination triplet, is covalently attached to a solid surface (see, Fig 20). Preferentially, transparent glass microscope slides coated by streptavidine are used and the DNA is attached via an 5'-biotin tag. Other method of DNA immobilization by photochemical or chemical methods are also feasible. The surface on which the DNA is attached is overlaid by a solution containing the complete in vitro translation system. In order to assure the immobilization of the "on spot" synthesized nascent polypeptide remains attached in the vicinity of the corresponding immobilized DNA matrix a C-terminal puromycin-dependent tagging system of the polypeptide is included in the bacterial in vitro translation extract. Biotin coupling the puromycin analogue is the obvious choice to achieve such immobilisation on streptavidin-coated solid plates. But alternative covalent coupling is also highly feasible. It was experimentally proven that puromycin analogue is also active in immobilized form provided the length of the linker to the solid surface is appropriate. It is, therefore, not important if the puromycin analogue carrying a biotin tag becomes immobilized prior to is attachment to the C-terminus of the protein. The "on spot" in vitro protein biosynthesis technology relies on selective depletion of a particular release factor from the translation extract, which is achieved by polyclonal antibodies against RF1 and RF2 in the case of UAG and UGA termination codons, respectively. Preferentially, polyclonal antibodies against orthologous release factors from Thermus thermophilus are used. The biotin is covalently attached to a puromycin analogue PupC5. Important feature of the system is that as a result of RF-depletion the biotin-puromycin, or chemically active puromycin analogue, are incorporated only into the full-length polypeptide. The transcription- translation coupling and the puromycin-analogue tagging of the protein will assure the "on spot" topospecific synthesis of the translation product on the two-dimensional solid support (see, Fig 20). It is a natural feature of the system that each immobilised DNA matrix will be exponentially multiplied by transcription and translation in close vicinity of the location on which the particular DNA sequence was attached. Alternatively, a protein array can be also constructed by microcompartmentisation of the DNA matrix on the solid support.
For control purposes the gene of the expressed protein can be coupled with a reporter gene linked via a cleavable protease site. Preferentially, the esterase 2 from Alicyclobacillus acidocaldarius, a single chain, thermostable enzyme is used as an sensitive reporter group, but other types of labelling using radioactive or spectroscopic reporter groups are possible. The described method is suitable for:
• in vitro preparation of protein microarrays without need of protein purification and posttranslational modification,
• preparation of solid surfaces specifically coated by polypeptides, enzymes or functionally active proteins,
• preparation of biosensors,
• preparation of coated surfaces for plasmon resonance experiments or atomic force microscopy measurements
• preparation of materials for affinity purification of biomacromolecules
• preparation of biocompatible and bioactive materials biotechnological applications, protetics and other medical applications. Example 18: In vitro selection and presentation of proteins by topospecific translation display (SPOT display)
A similar system as described above can be used for in vitro selection of proteins from a library of mutated DNA matrices. The PCR product that contains the modules depicted in Fig. 20. and carries a biotin tag, or a reactive group on the 5'-end. The coding part of the immobilised matrix DNA contains a random sequence. The correct sequence coding for the required protein is then selected. Many of the sequences combinations will cause terminations and synthesize short polypeptide. These sequences will be eliminated on the bases of missing esterase activity. The esterase reporter gene is located on the C-terminal site of the analysed protein, therefore each termination event in the analysed gene will result in inactive esterase. Enzymatic activity of the esterase can be detected with sensitivity better then 103 molecules/spot. DNA in the positive spots will be multiplied by PCR and 5'— biotinylated. After appropriate dilution the "Winner" DNA is placed on the new plate and the procedure is repeated. This can be repeated several times. In the next cycles a selection principle directed to a biochemical property of the N-terminaly placed protein ("active esterase" in Fig 20) is applied. Finally, the "winner" DNA sequences are cloned and sequenced.
The described SPOT-display method relies on following features:
• Immobilisation of DNA matrix on a solid support
• Proteins are coded by a few DNA molecules located on particular spot. This is achieved by maximal possible dilution of the DNA used for attachment to the solid support,
• Puromycin analogue is immobilized via an appropriate long linker to the same location on the solid support as is the DNA matrix,
• Immobilized Puromycin analogue is specifically incorporated in the full-length polypeptide in the position determined by stop codons, under condition of depletion of the release factors from the in vitro translation system.
The describe SPOT-display is suitable for protein engineering:
• Selection of enzymes with tailored enzymatic properties • Selection of polypeptides with tailored binding properties,
• Identification of optimal sequences for artificial antibodies.
Example 19: Esterase immobilization assay
The attachment of biotin to esterase was further demonstrated by immobilization of the corresponding cytidin(Biotin)-puromycin-protein conjugate (for formula see Fig. 4 and 5) directly from the translation mixture to the surface of streptavidin-coated polystyrene plates (Fig. 22 B, well 5). The streptavidin-coated well treated with C- terminally biotinylated esterase was enzymatically active (Fig. 22 B, well 5) whereas the control wells did not bind the polypeptide and had only low, residual esterase activity. A similar affinity of the in vitro biotinylated esterase to streptavidin was demonstrated for Streptavidin-Sepharose (Fig. 22 A). Some residual activity in the control experiments was probably due to unspecific adsorption of the esterase to the polymeric surface. Thus, it was demonstrated that in vitro preparation of cytidin(biotin)-puromycin-esterase conjugate does not alter its activity and can be used for enzyme or other protein immobilization.
For this Esterase immobilization assay 20 μl of translation mixture after 120 min of incubation (performed according to Fig. 10 A without radioactive labelling) were mixed with 5 μl of Streptavidin-Sepharose and incubated for 30 min. at 37°C. Then the matrix was spun down and supernatant was discarded. The Sepharose was washed three times with 0.5 ml of 150 mM NaCI in 20 mM Na-phosphate buffer pH 7.5 and mixed with 0.2 mM p-nitrophenyl acetate in 1 ml of 50 mM phosphate buffer pH 7.5. The production of p-nitrophenoxide was monitored at 405 nm in 1 cm path- length cells with UV-Spectral photometer DU 640 (Beckman, Fullerton, USA) at 25°C. Initial rates were calculated by linear least-square analysis of time courses comprising less than 10% of the total substrate turnover.
Binding of the biotinylated esterase to streptavidin coated polystyrene plates (Roche GmbH, Mannheim, Germany) was performed according to the manual provided by the supplier. 10μl of translation mixture after 120 min of incubation were incubated in a well for 30 min followed by three washing steps with 300 μl of of 150 mM NaCl in 20 mM Na-phosphate buffer pH 7.5. Activity staining of the esterase in the wells was performed as described (Higerd 1973 loc. cit.) with Fast Blue BB Salt and β-naphthyl- acetate. Example 20: Complementing the depletion of RF1
In order to improve C-terminal incorporation of puromycin, the RF1 responsible for termination at the UAA and the UAG stop codons was inactivated in the in vitro translation system from E. coli by rabbit antibodies raised against RF1 from Thermus thermophilus as described herein. As a template for cell free translation, plasmid DNA that encoded mRNA for synthesis of the esterase from Alicyclobacillus acidocaldarius (Manco 1998 loc. cit.) as described herein and terminated by UAG stop codon was used. The functionally active enzyme was produced up to a concentration of 200 μg/ml. Its C-terminal labeling was monitored by incorporation of [32P]pGpCpPuromycin (Fig. 23 1a) into the full-length protein (Fig. 21A). In accordance with the report of Yanagawa and coworkers (Miyamoto-Sato 2000 loc. cit.), the yield of puromycin derivative conjugation with the esterase was very low (approximately 6%) in the presence of endogenous E. coli RF1 (Fig. 21A, lane 0). Deactivation of RF1 with rabbit antibodies raised against T. thermophilus RF1 leads to a strong stimulation of [32P]pGpCpPuromycin incorporation into the C-terminus and provided a puromycin-conjugated esterase with more than 80% yield (Fig. 21B). The conjugation yield was determined as a ratio between the amount of active enzyme produced in the system that was calculated from esterase specific activity and the amount of puromycin derivative attached to the full length product calculated on the base of 32P radioactivity by the Phosphoimager software. The antibody concentration range in which the puromycin-mediated termination is turned on is very narrow (Fig. 21A and B) and correlates well with the concentration of RF1 at which an efficient incorporation is blocked via addition of purified E. coli RF1 (Fig. 21 C and D). Figure 21 shows that only at high anti RF1 antibody concentration [32P]pGpCpPuromycin starts to be incorporated efficiently into full-length esterase. Increase of antibody concentration above 2-3 μl of antiserum per 30 μl of translation mixture does not significantly improve the C-terminal incorporation of the drug. Inhibition of translation termination by anti RF1 specific antibodies possesses complete reversibility upon addition of purified E. coli RF1. The system supplied with 4 μl of antiserum per 30 μl of translation mixture was directly treated by exogenous RF1 at different concentrations. Thus, already at 20 nM of added RF1, incorporation of the [32P]pGpCpPuromycin into full-size esterase decreases (Fig. 21C), becoming undistinguishablθ from the antibody untreated system (Fig 21A, lane 0) at 0.5-5 μM RF1 range, where the yield of the reaction drops to the control system level (Fig. 21D). The effects caused by inactivation of RF1 by antibodies and restoration of its activity upon addition of purified E. coli RF1 into the system are specific in respect to full-length esterase. The formation of shorter polypeptides, that are probably products from truncated messengers, remains unaffected (compare Fig. 21 A and C). The in vitro transcription/translation and the incorporation of the puromycin derivative of this example was performed as described herein (e.g. Example 11). The synthesis and labelling of the puromycin derivative was performed as described in Example 8. Esterase activity assays were performed as described in Example 10.
Example 21 : Materials employed in this study
Within the present application, materials were purchased as follows. Taq polymerase was from Qiagen (Hilden, Germany), T4-DNA-I_igase from Promega (Mannheim, Germany), Factor Xa protease and restriction enzymes were from NewEngland Biolabs (Frankfurt, Germany). Fast Blue BB Salt, p-Nitrophenyl acetate and β-Naphthyl-acetate were from Fluka (Steinheim, Germany). 5-(and 6-) Carboxy- 2',7'-dichlorofluoresceine diacetate was from Molecular probes (Eugene, USA). Other analytical grade chemicals were obtained from Roth (Karlsruhe, Germany). Radioactive [14C]leucine (54 mCi/mmol) was from Amersham, Life Sciences (Freiburg, Germany).
The present invention refers to the following nucleotide and amino acid sequences:
SEQ lD No.1:
Nucleotide sequence encoding releasefactor 1 from Thermus thermophilus atgctggacaagcttgaccgcctagaggaagagtaccgggagctggaggc gctcctctccgacccggaggtgctgaaggacaaggggcgctaccagagcc tctcccgccgctacgccgagatgggggaggtgatcggcctcatccgggag taccggaaggtgctggaggacctggagcaggcggaaagccttcttgacga ccccgagctcaaggagatggccaaggcggagcgggaggccctcctcgccc gcaaggaggccctggagaaggagctggagcgccacctcctgcctaaggac cccatggacgaaagggacgccatcgtagagatccgggcggggacgggagg ggaggaggccgccctcttcgcccgcgaccttttcaacatgtacctccgct tcgccgaggagatgggctttgagacggaggtcctggactcccaccccacg gacctcgggggcttctccaaggtggtctttgaggtgcggggcccgggggc ctacggcaccttcaagtacgagagcggggtccaccgggtgcaacgggtgc ccgtcaccgagacccaggggcggatccacacctccaccgccacggtggcc gtcctccccaaggcggaggaggaggacttcgccctcaacatggacgagat ccgcattgacgtgatgcgggcctcggggcccggggggcagggggtgaaca ccacggactcggcggtgcgggtggtccacctgcccacggggatcatggtc acctgccaggactcccgcagccagatcaagaaccgggagaaggccctcat gatcctaagaagccgtctcctggagatgaagcgggcggaggaggcggaaa ggctccggaagacccgccttgcccagatcggcaccggggagcgctcggag aagatccgcacctacaacttcccccagtcccgggtcacggaccaccgcat cgggttcaccacccacgacctcgagggcgtcctctccggccacctgaccc ccatcctggaggcgctcaagcgggccgaccaggagcgccagctcgcggcg ctggcggaagggtga
SEQ ID No.2:
Amino acid sequence of release factor 1 from Thermus thermophilus
MLDKLDRLEEEYRELEALLSDPEVLKDKGRYQSLSRRYAEMGEVIGLIREYRKVLEDLEQAESLLDDPELKEMAK
DLGGFSKVVFEVRGPGAYGTFKYESGVHRVQRVPVTETQGRIHTSTATVAVLPKAEEEDFALNMDEIRIDVMRAS GPGGQGVNTTDSAVRVVHLPTGIMVTCQDSRSQIKNREKALMILRSRLLEMKRAEEAERLRKTRLAQIGTGERSE KIRTYNFPQSRVTDHRIGFTTHDLEGVLSGHLTPILEALKRADQERQLAALAEG*
SEQ ID No.3:
Nucleotide sequence encoding Peptide chain release factor 2 (RF-2). - Thermus thermophilus ttgcgcctcgcttcgcaatctgctatcctggtaaaggtatggacctggaa cgcctcgcgcaacgcctggaaggcctcagggggtatctttgacatccccc aaaaggaaacccgtctaaaagagctggagcggcgcctcgaggacccctcc ctctggaacgatcccgaggccgcccgcaaggtgagccaggaggccgcccg cctccggcgcaccgtggacaccttccgctccctggaaagcgacctccagg gccttttggagctcatggaggagcttcccgccgaggaacgggaggccctc aagcccgagctggaggaggccgcgaagaagctggacgagctctaccacca gaccctcctcaacttcccccacgcggagaagaacgccatcctcaccatcc agcccggggccgggggcacggaggcctgcgactgggcggagatgctccta aggatgtacacccgcttcgccgagcgccagggcttccaggtggaggtggt ggacctcacccctgggcccgaggcgggcattgactacgcccagatcctgg tcaagggggagaacgcctacggcctcctttcccccgaggccggggtgcac cgcctggtgcgcccttccccctttgacgcctcgggccgccgccacacctc cttcgccggggtggaggtgatccccgaggtggacgaggaggtggaggtgg tgctcaagcccgaggagctccgcattgacgtgatgcgggcctcggggccc gggggccagggggtgaacaccacggactcggcggtgcgggtggtccacct gcccacggggatcaccgtgacctgccagaccacgcggagccagatcaaga acaaggaactcgccctcaagatcctcaaggcccgcctctacgagctggag cggaagaagcgggaggaagagctcaaggccctgaggggcgaggtgcggcc catagagtggggaagccagatccggagctacgtcctggacaagaactacg tcaaggaccaccgcaccgggctcatgcgccacgacccggaaaacgtcctg gacggggacctcatggacctgatctgggcgggcctggagtggaaggcggg ccgccgccaggggacggaggaggtggaggcggagtag SEQ ID No. 4:
Q5SM01 Peptide chain releasefactor 2 - Thermus thermophilic HB8
MRLASQSAILVKVWTWNASRNAWKASGGIFDIPQKETRLKELERRLEDPSLWNDPEAARK VSQEAARLRRTVDTFRSLESDLQGLLELMEELPAEEREALKPELEEAAKKLDELYHQTLL NFPHAEKNAILTIQPGAGGTEACDWABMLLRMYTRFAERQGFQVEWDLTPGPEAGIDYA QILVKGENAYGLLSPEAGVHRLVRPSPFDASGRRHTSFAGVEVIPEVDEEVEWLKPEEL RIDVMRASGPGGQGVNTTDSAVRVVHLPTGITVTCQTTRSQIKNKELALKILKARLYELE RKKREEELKALRGEVRPIEWGSQIRSYVLDKNYVKDHRTGLMRHDPENVLDGDLMDLIWA GLEWKAGRRQGTEEVEAE
SEQ ID No.5:
Nucleotide sequence encoding Peptide chain release factor 1 (RF-1) - Escherichia coli, Escherichia coli 06, Escherichia coli 0157:1-17, and Shigella fiexneri. atgaagccttctatcgttgccaaactggaagccctgcatgaacgccatga agaagttcaggcgttgctgggtgacgcgcaaactatcgccgaccaggaac gttttcgcgcattatcacgcgaatatgcgcagttaagtgatgtttcgcgc tgttttaccgactggcaacaggttcaggaagatatcgaaaccgcacagat gatgctcgatgatcctgaaatgcgtgagatggcgcaggatgaactgcgcg aagctaaagaaaaaagcgagcaactggaacagcaattacaggttctgtta ctgccaaaagatcctgatgacgaacgtaacgccttcctcgaagtccgagc cggaaccggcggcgacgaagcggcgctgttcgcgggcgatctgttccgta tgtacagccgttatgccgaagcccgccgctggcgggtagaaatcatgagc gccagcgagggtgaacatggtggttataaagagatcatcgccaaaattag cggtgatggtgtgtatggtcgtctgaaatttgaatccggcggtcatcgcg tgcaacgtgttcctgctacggaatcgcagggtcgtattcatacttctgct tgtaccgttgcggtaatgccagaactgcctgacgcagaaCtgccggacat caacccagcagatttacgcattgatactttccgctcgtcaggggcgggtg gtcagcacgttaacaccaccggttcggcaattcgtattactcacttgccg accgggattgttgttgaatgtcaggacgaacgttcacaacataaaaacaa agctaaagcactttctgttctcggtgctcgcatccacgctgctgaaatgg caaaacgccaacaggccgaagcgtctacccgtcgtaacctgctggggagt ggcgatcgcagcgaccgtaaccgtacttacaacttcccgcaggggcgcgt taccgatcaccgcatcaacctgacgctctaccgcctggatgaagtgatgg aaggtaagctggatatgctgattgaaccgattatccaggaacatcaggcc gaccaactggcggcgttgtccgagcaggaataa
SEQ ID No.6:
>sp|P07011 |RF1_ECOLI Peptide chain release factor 1 (RF-1) - Escherichia coli, Escherichia coli 06,
Escherichia coli O157:H7, and Shigella fiexneri. MKPSIVAKLEALHERHEEVQALLGDAQTIADQERFRALSREYAQLSDVSRCFTDWQQVQEDIETAQMMLDDPEMR EMAQDELREAKEKSEQLEQQLQVLLLPKDPDDERNAFLEVRAGTGGDEAALFAGDLFRMYSRYAEARRWRVEIMS ASEGEHGGYKEIIAKISGDGVYGRLKFESGGHRVQRVPATESQGRIHTSACTVAVMPELPDAELPDINPADLRID TFRSSGAGGQHVNTTDSAIRITHLPTGIVVECQDERSQHKNKAKALSVLGARIHAAEMAKRQQAEASTRRNLLGS GDRSDRNRTYNFPQGRVTDHRINLTLYRLDEVMEGKLDMLIEPIIQEHQADQLAALSEQE*
SEQ ID No.7:
Nucleotide sequence encoding Peptide chain release factor 2 (RF-2) - Escherichia coli. atgtttgaaattaatccggtaaataatcgcattcaggacctcacggaacgctccgacgttcttagggggtatcttgactacgacgccaag aaagagcgtc tggaagaagtaaacgccgagctggaacagccggatgtctggaacgaacccgaacgcgcacaggcgctgggtaaagagcgttcctccctcg aagccgttgt cgacaccctcgaccaaatgaaacaggggctggaagatgtttctggtctgctggaactggctgtagaagctgacgacgaagaaacctttaa cgaagccgtt gctgaactcgacgccctggaagaaaaactggcgcagcttgagttccgccgtatgttctctggcgaatatgacagcgccgactgctacctc gatattcagg cggggtctggcggtacggaagcacaggactgggcgagcatgcttgagcgtatgtatctgcgctgggcagaatcgcgtggtttcaaaactg aaatcatcga agagtcggaaggtgaagtggcgggtattaaatccgtgacgatcaaaatctccggcgattacgcttacggctggctgcgtacagaaaccgg cgttcaccgc gtggtgcgtaaaagcccgtttgactccggcggtcgtcgccacacgtcgttcagctccgcgtttgtttatccggaagttgatgatgatatt gatatcgaaa tcaacccggcggatctgcgcattgacgtttatcgcacgtccggcgcgggcggtcagcacgttaaccgtaccgaatctgcggtgcgtatta cccacatccc gaccgggatcgtgacccagtgccagaacgaccgttcccagcacaagaacaaagatcaggccatgaagcagatgaaagcgaagctttatga actggagatg cagaagaaaaatgccgagaaacaggcgatggaagataacaaatccgacatcggctggggcagccagattcgttcttatgtccttgatgac tcccgcatta aagatctgcgcaccggggtagaaacccgcaacacgcaggccgtgctggacggcagcctggatcaatttatcgaagcaagtttgaaagcag ggttatga
SEQ ID No.8:
>sp|P07012|RF2_ECOLI Peptide chain release factor 2 (RF-2) - Escherichia coli.
MFEINPVNNRIQDLTERSDVLRGYLDYDAKKERLEEVNAELEQPDVWNEPERAQALGKERSSLEAVV
DTLDQMKQGLEDVSGLLELAVEADDEETFNEAVAELDALEEKLAQLEFRRMFSGEYDSADCYLDIQA
GSGGTEAQDWASMLERMYLRWAESRGFKTEIIEESEGEVAGIKSVTIKISGDYAYGWLRTETGVHRLV
RKSPFDSGGRRHTSFSSAFVYPEVDDDIDIEINPADLRIDVYRTSGAGGQHVNRTESAVRITHIPTGIVT QCQNDRSQHKNKDQAMKQMKAKLYELEMQKKNAEKQAMEDNKSDIGWGSQIRSYVLDDSRIKDLRT GVETRNTQAVLDGSLDQFIEASLKAGL
SEQ ID No. 9:
Nucleotide sequence encoding Peptide chain release factor homolog (RF-H) - Escherichia coli. atgggcattaagcgaaagtggtgtggcactattcagtggatttgtccgagtccgtatcggcctcatcatgggcgcaaaaactggtttctg ggcattgggc gttttaccgctgatgagcaggaacaatcggatgcaatccgttatgagacgctgcgttcgtcggggccgggcggtcaacatgtcaataaaa ccgactcggc ggtacgcgccacgcatttggcatccggtattagcgtgaaggttcagtcagagcgtagtcagcatgctaacaagcggctggcacgattgct gattgcctgg aagctggagcaacagcaacaggaaaatagcgcggcgctgaaatcgcagcggcgaatgttccatcaccagattgaacgtggcaacccgcga cggacattta cagggatggcttttatcgaaggataa
SEQ ID No.10:
>sp|P28369|RFH_ECOLI Peptide chain release factor homolog (RF-H) - Escherichia coli.
MGIKRKWCGTIQWICPSPYRPHHGRKNWFLGIGRFTADEQEQSDAIRYETLRSSGPGGQHVNKTDS
AVRATHLASGISVKVQSERSQHANKRLARLLIAWKLEQQQQENSAALKSQRRMFHHQIERGNPRRTF
TGMAFIEG
SEQ ID No. 11 :
Nucleotide sequence encoding Peptide chain release factor homolog - Escherichia coli06. atgatcttgctacaactctcctctgctcaggggccggaagaatgttgtctcgcagtgagaaaagcactggacaggctgattaaagaagct acccgacagg acgtcgcggtaacggtgctggaaacagaaacgggtcgctactctgacacactgcgttcggcgctgatttctctggatggcgataacgcat gggctctaag cgaaagctggtgcggcactattcagtggatttgtccgagtccgtatcggcctcatcatgggcgcaaaaactggtttctgggcattgggcg ttttaccgct gatgagcaggaacaatcggatgcaatccgttatgagacgctgcgttcgtcggggccgggcggtcaacatgtcaataaaaccgactcggcg gtacgcgcca cgcatctggcatccggtattagcgtgaaggttcagtcagagcgcagtcagcatgctaacaaacggctggcgcgattactgattgcctgga agctggaaca acagcaacaggaaaatagcgcggcgctgaaatcgcagcggcgaatgttccatcaccagattgaacgtggcaacccgcgacgaacgtttac agggatggcc tttatcgaagggtaa SEQ ID No. 12:
>tr|Q8FKM9 Peptide chain release factor homolog - Escherichia coli 06.
MILLQLSSAQGPEECCLAVRKALDRLIKEATRQDVAVTVLETETGRYSDTLRSALISLDGDNAWALSES
WCGTIQWICPSPYRPHHGRKNWFLGIGRFTADEQEQSDAIRYETLRSSGPGGQHVNKTDSAVRATH
LASGISVKVQSERSQHANKRLARLLIAWKLEQQQQENSAALKSQRRMFHHQIERGNPRRTFTGMAFI
EG
SEQ ID No. 13:
Nucleotide sequence encoding Probable peptide chain release factor - Escherichia coli O157:H7. gtgctggaaacagaaacgggccgctactctgacacgctgcgttcggcgctgatttctctggatggcgacaacgcatgggcgttaagcgaa agttggtgcg gcactattcagtggatttgtctgagtccgtatcggcctcatcatgggcgcaaaaactggtttctgggcattgggcgttttaccgctgatg agcaggaaca atcggatgcaatccgttatgagacgctgcgtccgtcggggccgggcggtcaacatgtcaataaaaccgactcggcggtacgcgccacgca tctggcaacc gggattagcgtgaaggttcagtcagaacgcagccagcatgctaacaaacggctggcgcgattgctgattgcctggaagctggagcagcag caacaggaaa atagcgcagtgctgaaatcgcagcggcgaatgttccatcaccagattgaacgtggcaacccgcgacgaacgttcacagggatggctttta tcgaaggata a
SEQ ID No.14:
>tr|Q8X7N9 Probable peptide chain release factor - Escherichia coli O157:H7.
MLETETGRYSDTLRSALISLDGDNAWALSESWCGTIQWICLSPYRPHHGRKNWFLGIGRFTADEQEQ
SDAIRYETLRPSGPGGQHVNKTDSAVRATHLATGISVKVQSERSQHANKRLARLLIAWKLEQQQQEN
SAVLKSQRRMFHHQIERGNPRRTFTGMAFIEG
SEQ ID No. 15
Nucleotide sequence encoding ERF1_RABIT Eukaryotic peptide chain release factor subunit 1 (eRF1)
(Eukaryotic release factor 1) - Oryctolagus cuniculus (Rabbit)
atggcggacgaccccagtgctgccgacaggaacgtggaaatctggaagatcaagaagctcattaagagcttggaggcggcccgcggcaat ggcaccagca tgatatcattgatcattcctcccaaagaccagatttcccgagtggcaaaaatgttagcagatgaatttggaactgcatccaacattaagt cacgagtaaa ccgcctttcagtcctgggagccattacatctgtacaacaaagactcaaactttataacaaagtacctccaaatggtctggttgtttactg tggaacaatt gtaacagaagaaggaaaggaaaagaaagtcaacattgactttgaacctttcaaaccaattaatacgtcattgtatttgtgtgacaacaaa ttccatacag aggctcttacagcactactttcagatgatagcaagtttggcttcattgtaatagatggtagtggtgcactttttggcacactgcagggaa atacaagaga agtcctgcacaaattcactgtggatctcccaaagaaacacggtagaggaggtcagtcagccttgcgttttgcccgtttaagaatggaaaa gcgacacaac tatgttcggaaagtagcagagactgctgtacagctgtttatttctggggacaaagtgaatgtggctggtctcgttttagctggatcagct gactttaaaa ctgaactaagtcaatctgatatgtttgaccagaggttgcaatcaaaagttttaaaattagttgatatatcctatggcggtgaaaatggat tcaaccaagc tattgagttatctactgaggtcctctccaacgtgaaattcattcaagagaagaaattaataggacgatactttgatgaaatcagtcaaga cacgggcaag tactgttttggagttgaagatacgctaaaagctttggaaatgggagccgtagaaattctaatagtctatgaaaatttggatataatgaga tacgttcttc attgccaaggcacagaagaggagaaaattctttacctaactccagaacaagagaaggataaatctcatttcacagacaaagagacaggac aggaacatga gctgattgagagcatgcccctgttggaatggtttgctaacaactataaaaaatttggagctacattggaaattgtcacagataagtcaca agaaggatcc cagtttgtgaaaggatttggtggaattggaggtatcttgcggtaccgagtagatttccagggaatggaatatcaaggaggagacgatgaa ttttttgacc ttgatgactactag
SEQ ID No.16:
>sp|P62497|ERF1_RABIT Eukaryotic peptide chain release factor subunit 1 (eRF1) (Eukaryotic release factor 1) - Oryctolagus cuniculus (Rabbit).
MADDPSAADRNVE)WKIKKLIKSLEAARGNGTSMISLIIPPKDQISRVAKMLADEFGTASNIKSRVNRLS
VLGAITSVQQRLKLYN KVPPNGLVVYCGTIVTEEGKEKKVNIDFEPFKPINTSLYLCDNKFHTEALTALL
SDDSKFGFIVIDGSGALFGTLQGNTREVLHKFTVDLPKKHGRGGQSALRFARLRMEKRHNYVRKVAE
TAVQLFISGDKVNVAGLVLAGSADFKTELSQSDMFDQRLQSKVLKLVDISYGGENGFNQAIELSTEVLS
NVKFIQEKKLIGRYFDEISQDTGKYCFGVEDTLKALEMGAVEILIVYENLDIMRYVLHCQGTEEEKILYLT
PEQEKDKSHFTDKETGQEHELIESMPLLEWFANNYKKFGATLEIVTDKSQEGSQFVKGFGGIGGILRY
RVDFQGMEYQGGDDEFFDLDDY
SEQ ID No. 17: Nucleotide sequence encoding Eukaryotic polypeptide chain release factor3 (Fragment) - Oryctolagus cuniculus (Rabbit) ctggcggcggcggccgaggcccagcgtgaccacctcagcgcggccttcagccggcagctcaacgtcaacgccaaacctttcgtgcccaac gtccacgccg cggagttcgtaccgtctttcctgcggggcccggccccgcctccagccccggctggcgccgccggcaacaaccacggagcgggcagcgtcg cgggaggccc ttcggcacctgtggaatcctctcaagaggaacagtcattgtgtgaaggctccatttcagctgttagcatggaactttcagaacctgttgt agagaacgga gagacagaaatgtccccagaagaatcatgggagcacaaagaagaaataagtgaggcagagccagggggtggctccctgggagatggaagg ccaccggagg aaggtgcccaagaaatgatggaggaggaagaggaaatgccaaagcccaaatctgtagctgcgcctcctggtgcccctaaaaaagaacatg taaatgtagt gtttattgggcatgtagatgctggcaagtcaaccattggaggccaaataatgtatttgactggaatggttgataaaaggacacttgagaa atatgaaaga gaagctaaagaaaaaaacagagaaacttggtacttgtcttgggccctagatacaaatcaggaagaacgagacaaaggtaaaacagtcgaa gtgggtcgtg cctactttgaaacagaaaagaagcatttcacaattctagacgcccctggccacaagagttttgtcccaaatatgattggtggcgcctctc aagctgattt ggctgtgctggtcatctctgccaggaaaggagagtttgaaactggatttgaaaaaggaggacagacaagagaacacgcaatgttggcaaa gacagcaggt gtaaagcacttaattgtgcttattaataagatggatgacccaacagtgaattggagcaacgagagatatgaagaatgtaaagagaaacta gtgccatttt tgaaaaaagttggcttcaatcccaaaaaggacattcactttatgccctgctcaggactgactggagcaaatctcaaagagcaatcagatt tctgtccttg gtacattggattaccatttattccatatctggataatttgccaaacttcaatagatcagttgatggaccaatcagactgccgattgtgga taagtacaag gatatgggcactgtggtcctgggaaagctggaatcgggatetatttgtaaaggccagcagctagtgatgatgccgaacaagcacaacgtg gaagttcttg gaatactttctgatgatgtagaaactgattctgtagccccaggtgagaacctgaaaatcagactcaaaggaattgaggaagaagagattc ttccaggatt catcctttgtgatcttaataatctttgccattctggacgcacatttgatgcccagatagtgattatagagcacaaatccatcatctgccc agggtacaat gcggtgctgcatattcatacctgtattgaggaagtcgagataacagccttaatctgcttggtagacaaaaagtcaggagagaaaagcaag actcggcccc gttttgtgaaacaagatcaagtgtgcattgcccgtGttcggacagcaggaaccatctgccttgagacctttaaggacttccctcagatgg gtcgttttac cttaagagatgagggtaagaccattgcaattggaaaagttctgaaactggttccagaaaaagactaa
SEQ ID No.18:
>tr|Q9N2G7 Eukaryotic polypeptide chain release factor 3 (Fragment) - Oryctolagus cuniculus
(Rabbit).
LAAAAEAQRDHLSAAFSRQLNVNAKPFVPNVHAAEFVPSFLRGPAPPPAPAGAAGNNHGAGSVAGG
PSAPVESSQEEQSLCEGSISAVSMELSEPWENGETEMSPEESWEHKEEISEAEPGGGSLGDGRPP
EEGAQEMMEEEEEMPKPKSVAAPPGAPKKEHVNVVFIGHVDAGKST)GGQIMYLTGMVDKRTLEKYE
REAKEKNRETWYLSWALDTNQEERDKGKTVEVGRAYFETEKKHFTILDAPGHKSFVPNMIGGASQAD
LAVLVISARKGEFETGFEKGGQTREHAMLAKTAGVKHLIVLINKMDDPTVNWSNERYEECKEKLVPFL
KKVGFNPKKDIHFMPCSGLTGANLKEQSDFCPWYIGLPFIPYLDNLPNFNRSVDGPIRLPIVDKYKDMG
TWLGKLESGSICKGQQLVMMPNKHNVEVLGILSDDVETDSVAPGENLKIRLKGIEEEEILPGFILCDLN
NLCHSGRTFDAQIVIIEHKSIICPGYNAVLHIHTCIEEVEITALICLVDKKSGEKSKTRPRFVKQDQVCIAR
LRTAGTICLETFKDFPQMGRFTLRDEGKTIAIGKVLKLVPEKD
SEQ ID No. 19
Nucleotide sequence encoding Eukaryotic peptide chain release factor subunit 1 (eRF1) (Eukaryotic release factor 1) - Drosophila melanogaster(Fruitfly). atgtctggcgaggaaacgtctgccgatcgcaatgtcgagatctggaaaatcaagaagctcatcaagagcctggaaatggcccgcggcaat ggaaccagca tgatttctttgattattccgccaaaggatcaaatctcgcgcgtcagcaagatgttggccgatgagtttggaacggcgtcgaacatcaagt cgcgtgtaaa tcggttgtccgtcctcggtgccattacgtcggtacagcacagactcaaattatacaccaaagtgcctcccaacggtttggtcatctactg cggcacaata gtcacagaggagggcaaggagaagaaggtgaacatagactttgagccattcaagcccataaacacgtcgctctacctctgcgacaacaag ttccacacgg aggccctcactgccctgctcgccgacgacaacaaatttggattcatcgtgatggatggtaacggagcgctattcggtacccttcagggca acacgcgcga ggtgctccacaagttcaccgtcgatctgccgaagaagcacggtcgtggtggtcagtccgcccttcgtttcgcccgtctgcgtatggagaa gcgccacaac tacgtgcggaaggtcgccgaggtggccacccagctcttcatcacgaacgacaagcccaacattgccggactcatcctggctggtagtgcg gatttcaaga ctgagcttagtcagtctgatatgttcgatcctcgtttgcaatcaaaagtcatcaagctggtggacgtgtcgtatggtggggaaaacggtt ttaaccaggc cattgaactggcggccgaatcattgcagaacgttaaattcatacaggagaagaaactcattggtcgctactttgatgaaatttctcagga tactggcaaa tactgttttggagtggaggacactttgcgggcactggaacttggctctgtagagactctcatttgttgggagaacctggatattcaacgt tatgttctca agaatcatgccaactcgacgtcaacgacagtattacatttgacgcccgagcaggaaaaggacaagtcgcacttcactgacaaggagagcg gggtagaaat ggagctgattgagtctcagccgctgctggaatggctggcaaacaactacaaaatgttcggcgccacactggagattatcacggataagtc ccaggaagga agtcagttcgtgcgaggtttcggtggaatcggcggtatcttacgctacaaggtggatttccagagtatgcagctcgatgaattggacaat gatggcttcg atctagatgattactag
SEQ ID No.20:
>sp|Q9VPH7|ERF1_DROME Eukaryotic peptide chain release factor subunit 1 (eRF1) (Eukaryotic release factor 1) - Drosophila melanogaster (Fruit fly).
MSGEETSADRNVEIWKIKKLIKSLEMARGNGTSMISLIIPPKDQISRVSKMLADEFGTASNIKSRVNRLS
VLGAITSVQHRLKL YTKVPPNGLVIYCGTIVTEEGKEKKVNIDFEPFKPINTSLYLCDNKFHTEALT ALLA
DDNKFGFIVMDGNGALFGTLQGNTREVLHKFTVDLPKKHGRGGQSALRFARLRMEKRHNYVRKVAE
VATQLFITNDKPNIAGLILAGSADFKTELSQSDMFDPRLQSKVIKLVDVSYGGENGFNQAIELAAESLQN
VKFIQEKKLIGRYFDEISQDTGKYCFGVEDTLRALELGSVETLICWENLDIQRYVLKNHANSTSTTVLHL
TPEQEKDKSHFTDKESGVEMELIESQPLLEWLANNYKMFGATLEIITDKSQEGSQFVRGFGGIGGILRY
KVDFQSMQLDELDNDGFDLDDY
SEQ ID No.21:
Nucleotide sequence encoding Eukaryotic peptide chain release factor subunit 1 (eRF1) (Eukaryotic release factor 1) (Omnipotent suppressor protein 1) - Saccharomyces cerevisiae (Baker's yeast). atggataacgaggttgaaaaaaatattgagatctggaaggtcaagaagttggtccaatctttagaaaaagctagaggtaatggtacttct atgatttcct tagttattcctcctaagggtctaattccactgtaccaaaaaatgttaacagatgaatatggtactgcctcgaatattaaatctagggtta atcgtctttc cgttttatctgctatcacttccacccaacaaaagttgaagctatataatactttgcccaagaacggtttagttttatattgtggtgatat catcactgaa gatggtaaagaaaaaaaggtcacttttgacatcgaaccttacaaacctatcaacacatccttatatttgtgtgataacaaatttcataca gaagttcttt cggaattgcttcaagctgacgacaagttcggttttatagtcatggacggtcaaggtactttgtttggttctgtgtccggtaatacgagaa ctgttttaca taaatttactgtcgatctgccaaaaaagcatggtagaggtggtcaatctgcgcttcgttttgctcgtttaagagaagaaaaaagacataa ttatgtgaga aaggtcgccgaagttgctgttcaaaattttattactaatgacaaagtcaatgttaagggtttaattttagctggttctgctgactttaag accgatttgg ctaaatctgaattattcgatccaagactagcatgtaaggttatttccatcgtggatgtttcttatggtggtgaaaacggtttcaaccagg ctatcgaact ttctgccgaagcgttggccaatgtcaagtatgttcaagaaaagaaattattggaggcatattttgacgaaatttcccaggacactggtaa attctgttat ggtatagatgatactttaaaggcattggatttaggtgcagtcgaaaaattaattgttttcgaaaatttggaaactatcagatatacattt aaagatgccg aggataatgaggttataaaattcgctgaaccagaagccaaggacaagtcgtttgctattgacaaagctaccggccaagaaatggacgttg tctccgaaga acctttaattgaatggctagcagctaactacaaaaacttcggtgctaccttggaattcatcacagacaaatcttcagaaggtgcccaatt tgtcacaggt tttggtggtattggtgccatgctgcgttacaaagttaattttgaacaactagttgatgaatctgaggatgaatattatgacgaagatgaa ggatccgact atgatttcatttaa
SEQ ID No.22:
>sp|P12385|ERF1_YEAST Eukaryotic peptide chain release factor subunit 1 (eRF1) (Eukaryotic release factor 1) (Omnipotent suppressor protein 1) - Saccharomyces cerevisiae (Baker's yeast).
MDNEVEKNIEIWKVKKLVQSLEKARGNGTSMISLVIPPKGLIPLYQKMLTDEYGTASNIKSRVNRLSVLS
AITSTQQKLKLYNTLPKNGLVLYCGDIITEDGKEKKVTFDIEPYKPINTSLYLCDNKFHTEVLSELLQADD
KFGFIVMDGQGTLFGSVSGNTRTVLHKFTVDLPKKHGRGGQSALRFARLREEKRHNYVRKVAEVAV
QNFITNDKVNVKGLILAGSADFKTDLAKSELFDPRLACKVISIVDVSYGGENGFNQAIELSAEALANVKY
VQEKKLLEAYFDEISQDTGKFCYGIDDTLKALDLGAVEKLIVFENLETIRYTFKDAEDNEVIKFAEPEAKD
KSFAIDKATGQEMDWSEEPLIEWLAANYKNFGATLEFITDKSSEGAQFVTGFGGIGAMLRYKVNFEQ
LVDESEDEYYDEDEGSDYDFI
SEQ ID No. 23:
Nucleotide sequence encoding Eukaryotic peptide chain release factor GTP-binding subunit (ERF2) (Translation release factor 3) (ERF3) (ERF-3) (Omnipotent suppressor protein 2) (G1 to S phase transition protein 1) - Saccharomyces cerevisiae (Baker's yeast). atgtcggattcaaaccaaggcaacaatcagcaaaactaccagcaatacagccagaacggtaaccaacaacaaggtaacaacagataccaa ggttatcaag cttacaatgctcaagcccaacctgcaggtgggtactaccaaaattaccaaggttattctgggtaccaacaaggtggctatcaacagtaca atcccgacgc cggttaccagcaacagtataatcctcaaggaggctatcaacagtacaatcctcaaggcggttatcagcagcaattcaatccacaaggtgg ccgtggaaat tacaaaaacttcaactacaataacaatttgcaaggatatcaagctggtttccaaccacagtctcaaggtatgtctttgaacgactttcaa aagcaacaaa agcaggccgctcccaaaccaaagaagactttgaagcttgtctccagttccggtatcaagttggccaatgctaccaagaaggttggcacaa aacctgccga atctgataagaaagaggaagagaagtctgctgaaaccaaagaaccaactaaagagccaacaaaggtcgaagaaccagttaaaaaggagga gaaaccagtc cagactgaagaaaagacggaggaaaaatcggaacttccaaaggtagaagaccttaaaatctctgaatcaacacataataccaacaatgcc aatgttacca gtgctgatgccttgatcaaggaacaggaagaagaagtggatgacgaagttgttaacgatatgtttggtggtaaagatcacgtttctttaa ttttcatggg tcatgttgatgccggtaaatctactatgggtggtaatctactatacttgactggctctgtggataagagaactattgagaaatatgaaag agaagccaag gatgcaggcagacaaggttggtacttgtcatgggtcatggataccaacaaagaagaaagaaatgatggtaagactatcgaagttggtaag gcctactttg aaactgaaaaaaggcgttataccatattggatgctcctggtcataaaatgtacgtttccgagatgatcggtggtgcttctcaagctgatg ttggtgtttt ggtcatttccgccagaaagggtgagtacgaaaccggttttgagagaggtggtcaaactcgtgaacacgccctattggccaagacccaagg tgttaataag atggttgtcgtcgtaaataagatggatgacccaaccgttaactggtctaaggaacgttacgaccaatgtgtgagtaatgtcagcaatttc ttgagagcaa ttggttacaacattaagacagacgttgtatttatgccagtatccggctacagtggtgcaaatttgaaagatcacgtagatccaaaagaat gcccatggta caccggcccaactctgttagaatatctggatacaatgaaccacgtcgaccgtcacatcaatgctccattcatgttgcctattgccgctaa gatgaaggat ctaggtaccatcgttgaaggtaaaattgaatccggtcatatcaaaaagggtcaatccaccctactgatgcctaacaaaaccgctgtggaa attcaaaata tttacaacgaaactgaaaatgaagttgatatggctatgtgtggtgagcaagttaaactaagaatcaaaggtgttgaagaagaagacattt caccaggttt tgtactaacatcgccaaagaaccctatcaagagtgttaccaagtttgtagctcaaattgctattgtagaattaaaatctatcatagcagc cggtttttca tgtgttatgcatgttcatacagcaattgaagaggtacatattgttaagttattgcacaaattagaaaagggtaccaaccgtaagtcaaag aaaccacctg cttttgctaagaagggtatgaaggtcatcgctgttttagaaactgaagctccagtttgtgtggaaacttaccaagattaccctcaattag gtagattcac tttgagagatcaaggtaccacaatagcaattggtaaaattgttaaaattgccgagtaa
SEQ ID No.24:
>sp|P05453|ERF2_YEAST Eukaryotic peptide chain release factor GTP-binding subunit (ERF2) (Translation release factor 3) (ERF3) (ERF-3) (Omnipotent suppressor protein 2) (G1 to S phase transition protein 1) - Saccharomyces cerevisiae (Baker's yeast).
MSDSNQGNNQQNYQQYSQNGNQQQGNNRYQGYQAYNAQAQPAGGYYQNYQGYSGYQQGGYQ
QYNPDAGYQQQYNPQGGYQQYNPQGGYQQQFNPQGGRGNYKNFNYNNNLQGYQAGFQPQSQG
MSLNDFQKQQKQAAPKPKKTLKLVSSSGIKLANATKKVGTKPAESDKKEEEKSAETKEPTKEPTKVEE
PVKKEEKPVQTEEKTEEKSELPKVEDLKISESTHNTNNANVTSADALIKEQEEEVDDEWNDMFGGKD
HVSLIFMGHVDAGKSTMGGNLLYLTGSVDKRTIEKYEREAKDAGRQGWYLSWVMDTNKEERNDGKT
LEVGKAYFETEKRRYTILDAPGHKMYVSEMIGGASQADVGVLVISARKGEYETGFERGGQTREHALLA
KTQGVNKMWWNKMDDPTVNWSKERYDQCVSNVSNFLRAIGYNIKTDVVFMPVSGYSGANLKDHV
DPKECPWYTGPTLLEYLDTMNHVDRHINAPFMLPIAAKMKDLGTIVEGKIESGHIKKGQSTLLMPNKTA
VEIQNIYNETENEVDMAMCGEQVKLRIKGVEEEDISPGFVLTSPKNPIKSVTKFVAQIAIVELKSIIAAGF
SCVMHVHTAIEEVHIVKLLHKLEKGTNRKSKKPPAFAKKGMKVIAVLETEAPVCVETYQDYPQLGRFTL
RDQGTTIAIGKIVKIAE
SEQ ID No. 25:
Nucleotide sequence encoding Peptide chain release factor 1, mitochondrial precursor (MRF-1) -
Saccharomyces cerevisiae (Baker's yeast). atgtggctttcaaagttccagttcccctcgaggtccatctttaagggtgtttttttgggtcacaagttgccgttactagtgcgactgaca tcaacaacga ctaactctaagagcaacggcagcatcccaacacaatacacagaactctctccgttacttgttaagcaagcggaaaagtatgaagctgaac taaaagatct tgacaaagacctttcttgcggcattcattttgacgtgaataagcagaaacattatgctaaattatcagcgctcactgatacatttattga gtataaggaa aaactaaatgaattgaaaagtttacaagaaatgattgtatctgatccatcattaagggcggaggctgaacaagaatatgcagaactggtc ccccaatatg aaacgacctcctcaagattggtaaataaacttcttccaccgcatccgttcgcagataaacctagcttactagagcttcgaccgggtgtag gtggcattga agccatgatttttacccagaatctattggatatgtatattggctatgccaactatagaaaatggaagtatcgaattatatcgaaaaatga aaacgaaagt gggtcaggcattatcgatgccattctgagcattgaagaagccggatcttacgatcgtttgaggtttgaagcaggcgttcacagggtgcag agaattccta gtacggagactaaaggaaggacgcacacatccactgctgccgtggttgtgttacctcaaattggtgacgaatctgctaaatctattgacg cttatgaaag aacatttaaacctggtgaaatcagagttgacatcatgcgtgcaagcgggaaaggtggtcaacatgtgaatacgacagactctgctgtcag attaacGcat atcccctctggaattgttgtttccatgcaagatgaaagatctcaacacaagaataaggctaaagcatttacgatcttaagagcgagactt gcagagaagg aaaggctggaaaaggaggaaaaagaaagaaaagcaagaaagagtcaggtttctagcaccaataggtctgataaaattagaacatacaatt ttccacagaa cagaatcactgatcataggtgcgggttcacattgttggacctgccaggtgtgttatctggagaacgattggacgaagttattgaggcaat gtctaaatat gatagcaccgaacgggcaaaagaattattggagagtaactga
SEQ ID No.26:
>sp|P30775|RF1 M-YEAST Peptide chain release factor 1 , mitochondrial precursor (MRF-1) -
Saccharomyces cerevisiae (Baker's yeast).
MWLSKFQFPSRSIFKGVFLGHKLPLLVRLTSTTTNSKSNGSIPTQYTELSPLLVKQAEKYEAELKDLDK
DLSCGIHFDVNKQKHYAKLSALTDTFIEYKEKLNELKSLQEMIVSDPSLRAEAEQEYAELVPQYETTSS
RLVNKLLPPHPFADKPSLLELRPGVGGIEAMIFTQNLLDMYIGYANYRKWKYRIISKNENESGSGIIDAIL
SIEEAGSYDRLRFEAGVHRVQRIPSTETKGRTHTSTAAWVLPQIGDESAKSIDAYERTFKPGEIRVDIM
RASGKGGQHVNTTDSAVRLTHIPSGIWSMQDERSQHKNKAKAFTILRARLAEKERLEKEEKERKARK
SQVSSTNRSDKIRTYNFPQNRITDHRCGFTLLDLPGVLSGERLDEVIEAMSKYDSTERAKELLESN
SEQ ID No. 27:
Nucleotide sequence encoding Translation termination factor SUP35 (Fragment) - Saccharomyces cerevisiae (Baker's yeast). caacaatcagcaaaactaccagcaatacagccagaacggtaaccaacaacaaggtaacaacagataccaaggttatcaagcttacaatgc tcaagcccaa cctgcaggtgggtactaccaaaattaccaaggttattctgggtaccaacaaggtggctatcaacagtacaatcccgatgccggttaccag caacagtata atcctcaaggaggctatcaacagtacaatcctcaaggcggttatcagcagcaattcaatccacaaggtggccgtggaaattacaaaaact tcaactacaa taacagtttgcaaggatatcaagctggtttccaaccacagtctcaaggtatgtctttgaacgactttcaaaagcaacaaaagcaggccgc tcccaaacca aagaagactttgaagcttgtctccagttccggtatcaagttggccaatgctaccaagaaggttgacacaaaacctgccgaatctgaaaag aaagaggaag agaagtctgctgaaaccaaagaaccaactaaagagccaacaaaggtcgaagaaccagttaaaaaggaggagaaaccagtccagactgaag aaaagaagga ggaaaaatcggaacttccaaaggtagaagacctcaaaatctctgaatcaacagataataccaacaatgccaatgttaccagtgctgatgc cttgatcaag gaacaggaagaagaagtggatgacgaagttgttaacgatatgtttggtggtaaagatcacgtttctttaattttcatgggtcatgttgat gccggtaaat ctactatgggtggtaatctactatacttgactggctctgtggataagagaactattgagaaatatgaaagagaagccaaggatgcaggca gacaaggttg gtacttgtcatgggtcatggataccaacaaagaagaaagaaatgatggtaagactatcgaagttggtaaggcctactttgaaactgaaaa aaggcgttat accatattggatgctcctggtcataaaatgtacgtttccgagatgatcggtggtgcttctcaagctgatgttggtgttttggtcatttcc gccagaaagg gtgagtacgaaaccggttttgagagaggtggtcaaactcgtgaacacgccctattggccaagacccaaggtgttaataagatggttgtcg tcgtaaataa gatggatgacccaaccgttaactggtctaaggaacgttacgaccaatgtgtgagtaatgtcagcaatttcttgagagcaattggttacaa cattaagaca gacgtt
SEQ ID No.28:
>tr|Q6Q7H Translation termination factor SUP35 (Fragment) - Saccharomyces cerevisiae (Baker's yeast).
NNQQNYQQYSQNGNQQQGNNRYQGYQAYNAQAQPAGGYYQNYQGYSGYQQGGYQQYNPDAGY
QQQYNPQGGYQQYNPQGGYQQQFNPQGGRGNYKNFNYNNSLQGYQAGFQPQSQGMSLNDFQK
QQKQAAPKPKKTLKLVSSSGIKLANATKKVDTKPAESEKKEEEKSAETKEPTKEPTKVEEPVKKEEKP
VQTEEKKEEKSELPKVEDLKISESTDNTNNANVTSADALIKEQEEEVDDEWNDMFGGKDHVSLIFMG
HVDAGKSTMGGNLLYLTGSVDKRTIEKYEREAKDAGRQGWYLSWVMDTNKEERNDGKTIEVGKAYF
ETEKRRYTILDAPGHKMYVSEMIGGASQADVGVLVISARKGEYETGFERGGQTREHALLAKTQGVNK
MVWVNKMDDPTVNWSKERYDQCVSNVSNFLRAIGYNIKTDV
SEQ ID No.29:
Nucleotide sequence encoding Translation termination factor SUP35 (Fragment) - Saccharomyces cerevisiae (Baker's yeast). caacaatcagcaaaactaccagcaatacagccagaacggtaaccaacaacaaggtaacaacagataccaaggttatcaagcttacaatgc tcaagcccaa cctgcaggtgggtactaccaaaattaccaaggttattctgggtaccaacaaggtggctatcaacagtacaatcccgacgccggttaccag caacagtata atcctcaaggaggctatcaacagtacaatcctcaaggcggttatcagcagcaattcaatccacaaggtggccgtggaaattacaaaaact tcaactacaa taacaatttgcaaggatatcaagctggtttccaaccacagtctcaaggtatgtctttgaacgactttcaaaagcaacaaaagcaggccgc tcccaaacca aagaagactttgaagcttgtctccagttccggtatcaagttggccaatgctaccaagaaggttgacacaaaacctgccgaatctgataag aaagaggaag agaagtctgctgaaaccaaagaaccaactagagagccaacaaaggtcgaagaaccagttaaaaaggaggagaaaccagtccagactgaag aaaagaagga ggaaaaatcggaacttccaaaggtagaagaccttaaaatctctgaatcaacacataataccaacaatgccaatgttaccagtgctgatgc cttgatcaag gaacaggaagaagaagtggatgacgaagttgttaacgatatgtttggtggtaaagatcacgtttctttaattttcatgggtcatgttgat gccggtaaat ctactatgggtggtaatctactatacttgactggctctgtggataagagaactattgagaaatatgaaagagaagccaaggatgcaggca gacaaggttg gtacttgtcatgggtcatggataccaacaaagaagaaagaaatgatggtaagactatcgaagttggtaaggcctactttgaaactgaaaa aaggcgttat accatattggatgctcctggtcataaaatgtacgtttccgagatgatcggtggtgcttctcaagctgatgttggtgttttggtcatttcc gccagaaagg gtgagtacgaaaccggttttgagagaggtggtcaaactcgtgaacacgccctattggccaagacccaaggtgttaataagatggttgtcg tcgtaaataa gatggatgacccaaccgttaactggtctaaggaacgttacgaccaatgtgtgagtaatgtcagcaatttcttgagagcaattggttacaa cattaagaca gacgtt
SEQ ID No.30:
>tr|Q6Q7l2 Translation termination factor SUP35 (Fragment) - Saccharomyces cerevisiae (Baker's yeast).
NNQQNYQQYSQNGNQQQGNNRYQGYQAYNAQAQPAGGYYQNYQGYSGYQQGGYQQYNPDAGY
QQQYNPQGGYQQYNPQGGYQQQFNPQGGRGNYKNFNYNNNLQGYQAGFQPQSQGMSLNDFQK
QQKQAAPKPKKTLKLVSSSGIKLANATKKVDTKPAESDKKEEEKSAETKEPTREPTKVEEPVKKEEKP
VQTEEKKEEKSELPKVEDLKISESTHNTNNANVTSADALIKEQEEEVDDEVVNDMFGGKDHVSLIFMG
HVDAGKSTMGGNLLYLTGSVDKRTIEKYEREAKDAGRQGWYLSWVMDTNKEERNDGKTIEVGKAYF
ETEKRRYTILDAPGHKMYVSEMIGGASQADVGVLVISARKGEYETGFERGGQTREHALLAKTQGVNK
MVWVNKMDDPTVNWSKERYDQCVSNVSNFLRAIGYNIKTDV
SEQ ID No. 31: Nucleotide sequence encoding Translation termination factor SUP35 (Fragment) - Saccharomyces cerevisiae (Baker's yeast). caacaatcagcaaaactaccagcaatacagccagaacggtaaccaacaacaaggtaacaacagataccaaggttatcaagcttacaatgc tcaagcccaa cctgcaggtgggtactaccaaaattaccaaggttattctgggtaccaacaaggtggctatcaacagtacaatcccgacgccggttaccag caacagtata atcctcaaggaggctatcaacagtacaatcctcaaggcggttatcagcagcaattcaatccacaaggtggccgtggaaattacaaaaact tcaactacaa taacaatttgcaaggatatcaagctggtttccaaccacagtctcaaggtatgtctttgaacgactttcaaaagcaacaaaagcaggccgc tcccaaacca aagaagactttgaagcttgtctccagttccggtatcaagttggccaatgctaccaagaaggttggcacaaaacctgccgaatctgataag aaagaggaag agaagtctgctgaaaccaaagaaccaactaaagagccaacaaaggtcgaagaaccagttaaaaaggaggagaaaccagtccagactgaag aaaagacgga ggaaaaatcggaacttccaaaggtagaagaccttaaaatctctgaatcaacacataataccaacaatgccaatgttaccagtgctgatgc cttgatcaag gaacaggaagaagaagtggatgacgaagttgttaacgatatgtttggtggtaaagatcacgtttctttaattttcatgggtcatgttgat gccggtaaat ctactatgggtggtaatctactatacttgactggctctgtggataagagaactattgagaaatatgaaagagaagccaaggatgcaggca gacaaggttg gtacttgtcatgggtcatggataccaacaaagaagaaagaaatgatggtaagactatcgaagttggtaaggcctactttgaaactgaaaa aaggcgttat accatattggatgctcctggtcataaaatgtacgtttccgagatgatcggtggtgcttctcaagctgatgttggtgttttggtcatttcc gccagaaagg gtgagtacgaaaccggttttgagagaggtggtcaaactcgtgaacacgccctattggccaagacccaaggtgttaataagatggttgtcg tcgtaaataa gatggatgacccaaccgttaactggtctaaggaacgttacgaccaatgtgtgagtaatgtcagcaatttcttgagagcaattggttacaa cattaagaca gacgtt
SEQ ID No.32:
>tr|Q6Q7l3 Translation termination factor SUP35 (Fragment) - Saccharomyces cerevisiae (Baker's yeast).
NNQQNYQQYSQNGNQQQGNNRYQGYQAYNAQAQPAGGYYQNYQGYSGYQQGGYQQYNPDAGY
QQQYNPQGGYQQYNPQGGYQQQFNPQGGRGNYKNFNYNNNLQGYQAGFQPQSQGMSLNDFQK
QQKQAAPKPKKTLKLVSSSGIKLANATKKVGTKPAESDKKEEEKSAETKEPTKEPTKVEEPVKKEEKP VQTEEKTEEKSELPKVEDLKISESTHNTNNANVTSADALIKEQEEEVDDEVVNDMFGGKDHVSLIFMG HVDAGKSTMGGNLLYLTGSVDKRTIEKYEREAKDAGRQGWYLSWVMDTNKEERNDGKTIEVGKAYF ETEKRRYTILDAPGHKMYVSEMIGGASQADVGVLVISARKGEYETGFERGGQTREHALLAKTQGVNK MVWVNKMDDPTVNWSKERYDQCVSNVSNFLRAIGYNIKTDV
SEQ ID No. 33:
Nucleotide sequence encoding Translation termination factor SUP35 (Fragment) - Saccharomyces cerevisiae (Baker's yeast). caacaatcagcaaaactaccagcaatacagccagaacggtaaccaacaacaaggtaacaacagataccaaggttatcaagcttacaatgc tcaagcccaa cctgcaggtgggtactaccaaaattaccaaggttattctgggtaccaacaaggtggctatcaacagtacaatcccgatgccggttaccag caacagtata atcctcaaggaggctatcaacagtacaatcctcaaggcggttatcagcagcaattcaatccacaaggtggccgtggaaattacaaaaact tcaactacaa taacagtttgcaaggatatcaagccggtttccaaccacagtctcaaggtatgtctttgaacgactttcaaaagcaacaaaagcaggccgc tcccaaacca aagaagactttgaagcttgtctccagttccggtatcaagttggccaatgctaccaagaaggttgacacaaaacctgccgaatctgaaaag aaagaggaag agaagtctgctgaaaccaaagaaccaactaaagagccaacaaaggtcgaagaaccagttaaaaaggaggagaaaccagtccagactgaag aaaagaagga ggaaaaatcggaacttccaaaggtagaagacctcaaaatctctgaatcaacacataataccaacaatgccaatgttaccagtgctgatgc cttgatcaag gaacaggaagaagaagtggatgacgaagttgttaacgatatgtttggtggtaaagatcacgtttctttaattttcatgggtcatgttgat gccggtaaat ctactatgggtggtaatctactatacttgactggctctgtggataagagaactattgagaaatatgaaagagaagccaaggatgcaggca gacaaggttg gtacttgtcatgggtcatggataccaacaaagaagaaagaaatgatggtaagactatcgaagttggtaaggcctactttgaaactgaaaa aaggcgttat accatattggatgctcctggtcataaaatgtacgtttccgagatgatcggtggtgcttctcaagctgatgttggtgttttggtcatttcc gccagaaagg gtgagtacgaaaccggttttgagagaggtggtcaaactcgtgaacacgccctattggccaagacccaaggtgttaataagatggttgtcg tcgtaaataa gatggatgacccaaccgttaactggtctaaggaacgttacgaccaatgtgtgagtaatgtcagcaatttcttgagagcaattggttacaa cattaagaca gacgtt SEQ ID No. 34:
>tr|Q6Q7l4 Translation termination factor SUP35 (Fragment) - Saccharomyces cerevisiae (Baker's yeast).
NNQQNYQQYSQNGNQQQGNNRYQGYQAYNAQAQPAGGYYQNYQGYSGYQQGGYQQYNPDAGY
QQQYNPQGGYQQYNPQGGYQQQFNPQGGRGNYKNFNYNNSLQGYQAGFQPQSQGMSLNDFQK
QQKQAAPKPKKTLKLVSSSGIKLANATKKVDTKPAESEKKEEEKSAETKEPTKEPTKVEEPVKKEEKP
VQTEEKKEEKSELPKVEDLKISESTHNTNNANVTSADALIKEQEEEVDDEVVNDMFGGKDHVSLIFMG
HVDAGKSTMGGNLLYLTGSVDKRTIEKYEREAKDAGRQGWYLSWVMDTNKEERNDGKTIEVGKAYF
ETEKRRYTILDAPGHKMYVSEMIGGASQADVGVLVISARKGEYETGFERGGQTREHALLAKTQGVNK
MVWVNKMDDPTVNWSKERYDQCVSNVSNFLRAIGYNIKTDV
SEQ ID No.35:
Nucleotide sequence encoding Translation termination factor SUP35 (Fragment) - Saccharomyces cerevisiae (Baker's yeast). caacaatcagcaaaactaccagcaatacagccagaacggtaaccaacaacaaggtaacaacagataccaaggttatcaagcttacaatgc tcaagcccaa cctgcaggtgggtactaccaaaattaccaaggttattctgggtaccaacaaggtggctatcaacagtacaatcccgacgccggttaccag caacagtata atcctcaaggaggctatcaacagtacaatcctcaaggcggttatcagcagcaattcaatccacaaggtggccgtggaaattacaaaaact tcaactacaa taacaatttgcaaggatatcaagctggtttccaaccacagtctcaaggtatgtctttgaacgactttcaaaagcaacaaaagcaggccgc tcccaaacca aagaagactttgaagcttgtctccagttccggtatcaagttggccaatgctaccaagaaggttgacacaaaacctgccgaatctgataag aaagaggaag agaagtctgctgaaaccaaagaaccaactaaagagccaacaaaggtcgaagaaccagttaaaaaggaggagaaaccagtccagactgaag aaaagacgga ggaaaaatcggaacttccaaaggtagaagaccttaaaatctctgaatcaacacataataccaacaatgccaatgttaccagtgctgatgc cttgatcaag gaacaggaagaagaagtggatgacgaagttgttaacgatatgtttggtggtaaagatcacgtttctttaattttcatgggtcatgttgat gccggtaaat ctactatgggtggtaatctactatacttgactggctctgtggataagagaactattgagaaatatgaaagagaagccaaggatgcaggca gacaaggttg gtacttgtcatgggtcatggataccaacaaagaagaaagaaatgatggtaagactatcgaagttggtaaggcctactttgaaactgaaaa aaggcgttat accatattggatgctcctggtcataaaatgtacgtttccgagatgatcggtggtgcttctcaagctgatgttggtgttttggtcatttcc gccagaaagg gtgagtacgaaaccggttttgagagaggtggtcaaactcgtgaacacgccctattggccaagacccaaggtgttaataagatggttgtcg tcgtaaataa gatggatgacccaaccgttaactggtctaaggaacgttacgaccaatgtgtgagtaatgtcagcaatttcttgagagcaattggttacaa cattaagaca gacgtt
SEQ ID No.36:
>tr|Q6Q7l6 Translation termination factor SUP35 (Fragment) - Saccharomyces cerevisiae (Baker's yeast).
NNQQNYQQYSQNGNQQQGNNRYQGYQAYNAQAQPAGGYYQNYQGYSGYQQGGYQQYNPDAGY
QQQYNPQGGYQQYNPQGGYQQQFNPQGGRGNYKNFNYNNNLQGYQAGFQPQSQGMSLNDFQK
QQKQAAPKPKKTLKLVSSSGIKLANATKKVDTKPAESDKKEEEKSAETKEPTKEPTKVEEPVKKEEKP
VQTEEKTEEKSELPKVEDLKISESTHNTNNANVTSADALIKEQEEEVDDEWNDMFGGKDHVSLIFMG
HVDAGKSTMGGNLLYLTGSVDKRTIEKYEREAKDAGRQGWYLSWVMDTNKEERNDGKTIEVGKAYF
ETEKRRYTILDAPGHKMYVSEMIGGASQADVGVLVISARKGEYETGFERGGQTREHALLAKTQGVNK
MVWVNKMDDPTVNWSKERYDQCVSNVSNFLRAIGYNIKTDV
SEQ ID No. 37:
Peptide chain release factor homolog (RF-H) - Escherichia coli.
MLETETGRYSDTLRSALVSLDGDNAWALSESWCGTIQWICPSPYRPHHGRKNWFLGIGRFTADEQE
QSDAIRYETLRSSGPGGQHVNKTDSAVRATHLASGISVKVQSERSQHANKRLARLLIAWKLEQQQQE
NSAALKSQRRMFHHQIERGNPRRTFTGMAFIEG
SEQ ID No. 38:
Nucleotide sequence encoding esterase 2 from Alicyclobacillus acidocaldarius:
1 ATGCCGCTCG ATCCCGTCAT TCAGCAGGTG CTCGATCAAC TCAACCGCAT
51 GCCTGCCCCG GACTACAAAC ATCTCTCCGC CCAGCAATTT CGTTCCCAAC
101 AGTCGCTGTT TCCTCCTGTC AAGAAGGAGC CCGTGGCCGA GGTCCGAGAG
151 TTTGACATGG ATCTGCCTGG CCGCACGCTC AAGGTGCGCA TGTACCGCCC
201 GGAGGGCGTC GAACCGCCCT ACCCCGCGCT CGTGTATTAT CACGGCGGCG
251 GTTGGGTCGT CGGAGACCTC GAGACGCACG ATCCCGTCTG CCGCGTCCTC
301 GCGAAAGACG GCCGCGCGGT CGTGTTCTCC GTCGACTACC GCCTGGCGCC
351 GGAGCACAAG TTCCCTGCCG CCGTGGAAGA CGCCTACGAC GCGCTTCAGT
401 GGATCGCGGA GCGCGCAGCG GACTTTCATC TCGATCCAGC CCGCATCGCG
451 GTCGGCGGAG ACAGCGCCGG AGGGAATCTT GCCGCTGTGA CGAGCATCCT 501 TGCCAAAGAG CGCGGCGGGC CGGCCATCGC GTTCCAGCTG CTCATCTACC
551 CTTCCACGGG GTACGATCCG GCTCATCCTC CCGCATCTAT CGAAGAAAAT
601 GCGGAAGGCT ATCTCCTGAC CGGCGGCATG ATGCTCTGGT TCCGGGATCA
651 ATACTTGAAC AGCCTGGAGG AACTCACGCA TCCGTGGTTT TCACCCGTCC
701 TCTACCCGGA CTTGAGCGGC TTGCCTCCGG CGTACATCGC GACGGCGCAG
751 TACGATCCGC TGCGCGACGT CGGCAAGCTT TACGCGGAAG CGCTGAACAA
801 GGCGGGCGTC AAGGTCGAGA TCGAGAACTT CGAAGATCTG ATCCACGGAT
851 TCGCACAGTT TTACAGCCTT TCGCCTGGCG CGACGAAGGC GCTCGTCCGC
901 ATTGCGGAGA AACTTCGAGA CGCGCTGGCC TGA
SEQ ID No.39:
Amino acid sequence of esterase 2 from Alicyclobacillus acidocaldarius:
1 MPLDPVIQQV LDQLNRMPAP DYKHLSAQQF RSQQSLFPPV KKEPVAEVRE 51 FDXDLPGRTL KVRXYRPEGV EPPYPALVYY HGGGWVVGDL ETHDPVCRVL ioi AKDGRAVVFS VDYRLAPEHK FPAAVEDAYD ALQWIAERAA DFHLDPARIA 151 VGGDSAGGNL AAVTSILAKE RGGPALAFQL LIYPSTGYDP AHPPASIEEN
201 AEGYLLTGGX XLWFRDQYLN SLEELTHPWF SPVLYPDLSG LPPAYIATAQ 251 YDPLRDVGKL YAEALNKAGV KVEIENFEDL IHGFAQFYSL SPGATKALVR 301 IAEKLRDALA
With respect to SEQ ID NO: 39, it is preferred that the amino acid residues indicated with X (position 53, 64, 210 and 211) are methionine residues (Met (M)) as encoded by the corresponding codon triplet "ATG" as shown in SEQ ID NO:1. The amino acid sequence corresponding to SEQ ID NO: 39 and having methionine residues (Met (M)) at amino acid position 53, 64, 210 and 211 is shown in SEQ ID NO: 55 of the sequence listing.
Sequences were retrieved from www.expasy.ch with ">sp|" indicating entry from swiss prot database and ">tr|" entry from TrEMBL database.

Claims

1. A cell-free translation system comprising a nonsense-codon suppressing agent and an anti-release factor antibody which precipitates and/or crosslinks a release factor in said cell-free translation system.
2. The cell-free translation system according to claim 1, wherein said cell-free translation system is a cell-free coupled transcription/translation system.
3. The cell-free translation system according to claim 1 or 2, wherein said cell- free translation system is of prokaryotic and/or eukaryotic origin.
4. The cell-free translation system according to any one of claims 1 to 3, wherein said cell-free translation system is of prokaryotic origin.
5. The cell-free translation system according to any one of claims 1 to 3, wherein said cell-free translation system is of eukaryotic origin.
6. The cell-free translation system according to any one of claims 1 to 4, wherein said cell-free translation system is an E. coli cell-free translation system.
7. The cell-free translation system according to any one of claims 1 to 3 and 5, wherein said cell-free translation system is a wheat germ extract cell-free translation system or a rabbit reticulocyte lysate cell-free translation system.
8. The cell-free translation system according to any one of claims 1 to 7, wherein said cell-free translation system comprises:
- a cell-free extract;
- ribonucleotide triphosphates;
- a RNA polymerase;
- magnesium ions;
- a template plasmid; - free amino acids; and/or
- aminoacyl-tRNAs.
9. The cell-free translation system according to any one of claims 1 to 8, wherein said cell-free translation system (further) comprises a radioactively labelled amino acid.
10. The cell-free translation system according to any one of claims 1 to 8, wherein said cell-free translation system (further) comprises [14C]leucine, [14C]valine and/or [14C]isoleucine.
11. The cell-free translation system according to any one of claims 1 to 10, wherein said nonsense-codon suppressing agent is puromycin or a derivative thereof and/or a suppressor tRNA.
12. The cell-free translation system according to any one of claims 1 to 11 , wherein said nonsense-codon suppressing agent is puromycin or a derivative thereof.
13. The cell-free translation system according to any one of claims 1 to 12, wherein said nonsense-codon suppressing agent is selected from the group consisting of:
(a) Puromycin;
(b) 5'-OH-CpPuromycin;
(c) 5'-OH-CpCpPuromycin;
(d) a puromycin derivative as defined in (a) to (c) having a residue covalently attached directly or via a linker to its 5'-position;
(e) a puromycin derivative as defined in (a) to (d) having a residue covalently attached directly or via a linker to the element N4 of the cytosine-residue of an 5' attached cytidine-residue; and (f) a puromycin derivative as defined in (a) to (e) having a residue covalently attached directly or via a linker to the element C5 of the cytosine-residue of an 5' attached cytidine-residue.
14. The cell-free translation system according to any one of claims 1 to 13, wherein said nonsense-codon suppressing agent comprises a puromycin derivative as defined in claim 13(a) to (d) having a residue covalently attached directly or via a linker to the element N4 of the cytosine-residue of an 5' attached cytidine-residue.
15. The cell-free translation system according to claim 13 or 14, wherein said residue is selected from the group consisting of DNA, RNA, locked DNA, PNA, oligonucleotide-thiophosphates, substituted ribo-oligonucleotides, and proteins.
16. The cell-free translation system according to claims 13 or 14, wherein said residue is selected from the group consisting of a fluorosphore (like Cy3), biotin or an other affinity tag, a reactive group for affinity labelling or any other reporter group.
17. The cell-free translation system according to any one of claims 13 to 16, wherein said linker is an aliphatic amine derivative.
18. The cell-free translation system according to any one of claims 1 to 17, wherein said nonsense-codon suppressing agent is selected from the group consisting of:
(a) 5'-OH-GpCpPuromycin;
(b) 5'-OH-GpCpCpPuromycin;
(c) 5'-OH-GpApCpCpPuromycin;
(d) 5'-OH-GpCpApCpCpPuromycin;
(e) 5'-OH-GpCpCpApCpCpPuromycin;
Figure imgf000115_0001
Figure imgf000116_0001
Figure imgf000117_0001
Figure imgf000118_0001
19. The cell-free translation system according to any one of claims 1 to 11 , wherein said nonsense-codon suppressing agent is suppressor tRNA.
20. The cell-free translation system according to any one of claims 1 to 11 and 19, wherein said nonsense-codon suppressing agent is suppressor tRNASer(CUA).
21. The cell-free translation system according to any one of claims 1 to 20, wherein said release factor is of prokaryotic or eukaryotic origin.
22. The cell-free translation system according to any one of claims 1 to 21 , wherein said release factor is of prokaryotic origin.
23. The cell-free translation system according to any one of claims 1 to 21, wherein said release factor is of eukaryotic origin.
24. The cell-free translation system according to any one of claim 1 to 22, wherein said release factor is from Thermus thermophilus or E. coli.
25. The cell-free translation system according to any one of claims 1 to 22 and 24, wherein said release factor is from Thermus thermophilus.
26. The cell-free translation system according to any one of claims 1 to 22, 24 and 25, wherein said release factor is the release factor 1 from Thermus thermophilus.
27. The cell-free translation system according to any one of claims 1 to 22 and 24, wherein said release factor is from E. coli.
28. The cell-free translation system according to any one of claims 1 to 22, 24 and 27, wherein said release factor is the release factor 1 , the release factor 2, the release factor homolog 1, the release factor homolog 2, the release factor homolog 3 or the release factor homolog 4 from E. coli.
29. The cell-free translation system according to any one of claims 1 to 22, 24, 27 and 28, wherein said release factor is the release factor 1 from E. coli.
30. The cell-free translation system according to any one of claim 1 to 21 and 23, wherein said release factor is from rabbit, fruit fly or yeast.
31. The cell-free translation system according to any one of claims 1 to 21 , 23 and 30, wherein said release factor is from rabbit.
32. The cell-free translation system according to any one of claims 1 to 21, 23, 30 and 31 , wherein said release factor is the release factor 1 or the release factor 3 from rabbit.
33. The cell-free translation system according to any one of claims 1 to 21, 23 and 30, wherein said release factor is from fruit fly.
34. The cell-free translation system according to any one of claims 1 to 21 , 23, 30 and 33, wherein said release factor is the release factor 1 from fruit fly.
35. The cell-free translation system according to any one of claims 1 to 21, 23 and 30, wherein said release factor is from yeast.
36. The cell-free translation system according to any one of claims 1 to 21 , 23, 30 and 35 wherein said release factor is the release factor 1 or the peptide chain release factor 1 from yeast.
37. The cell-free translation system according to any one of claims 1 to 36, wherein said release factor is selected from the group consisting of:
(a) a release factor encoded by a nucleotide sequence comprising a nucleotide sequence as shown in any one of SEQ ID NOS: 1; 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33 and 35;
(b) a release factor encoded by a nucleotide sequence coding for a polypeptide comprising an amino acid sequence as shown in any one of SEQ ID NOS: 2; 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 and 37;
(c) a release factor which is encoded by a nucleotide sequence of a nucleic acid molecule that hybridizes to the complement strand of a nucleic acid molecule comprising an nucleotide sequence as defined in (a) or (b) and which releases a translation product from a ribosome in a cell-free translation system;
(d) a release factor which comprises an amino acid sequence as shown in any one of SEQ ID NOS: 2; 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 and 37;
(e) a release factor which comprises an amino acid sequence which is at least 40% identical to the full length amino acid sequence as shown in any one of SEQ ID NOS: 2; 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 and 37; and
(f) a release factor encoded by a nucleotide sequence which is degenerated to a nucleotide sequence as defined in any one of (a) to (c).
38. The cell-free translation system according to any one of claims 1 to 37, wherein said anti-release factor antibody is directed against a release factor as defined in any one of claims 21 to 37.
39. The cell-free translation system according to any one of claims 1 to 38, wherein said anti-release factor antibody is monoclonal or polyclonal.
40. The cell-free translation system according to any one of claims 1 to 39, wherein said anti-release factor antibody is polyclonal.
41. The cell-free translation system according to any one of claims 1 to 40, wherein said anti-release factor antibody is provided in a serum.
42. The cell-free translation system according to any one of claims 1 to 40, wherein said anti-release factor antibody is purified.
43. The cell-free translation system according to any one of claims 1-4, 6, 8-22, 27-29 and 38-42, wherein said cell-free translation system is an E. coli cell- free translation system, said anti-release factor antibody precipitating and/or crosslinking a release factor is an antibody directed against the release factor 1 from Thermυs thermophilus and said release factor is release factor 1 from E. coli.
44. A kit comprising a cell free translation system as defined in any one of claims 1 to 43.
45. An anti-release factor antibody obtainable by
(a) eliciting an in vivo humoral response against release factor 1 from Thermus thermophilus or a fragment thereof in a non-human vertebrate; and
(b) obtaining a serum from said non-human vertebrate.
46. The anti-release factor antibody according to claim 45, wherein said release factor 1 from Thermus thermophilus or said fragment thereof is purified.
47. The anti-release factor antibody according to claim 45 or 46, wherein said anti- release factor antibody as obtained in the eliciting step (a) is purified.
48. The anti-release factor antibody according to any one of claims 45 to 47, wherein said release factor 1 from Thermus thermophilus or said fragment thereof is recombinantly produced.
49. The anti-release factor antibody according to any one of claims 45 to 48, wherein said release factor 1 from Thermus thermophilus or said fragment thereof is expressed in E. coli.
50. The anti-release factor antibody according to any one of claims 45 to 49, wherein said release factor 1 from Thermus thermophilus or said fragment thereof is purified and, wherein said purification comprises a heat-treatment.
51. The anti-release factor antibody according to claim 50, wherein said heat- treatment is at 65°C for 15 minutes.
52. The anti-release factor antibody according to any one of claims 45 to 51 , wherein said non-human vertebrate is selected from the group consisting of rat, mouse, rabbit, chicken, sheep, horse, goat, pig and donkey.
53. Use of an anti-release factor antibody according to any one of claims 45 to 52 for the preparation of a cell-free translation system.
54. A method for the production of an anti-release factor antibody comprising the steps of
(a) eliciting an in vivo humoral response against release factor 1 from Thermus thermophilus or a fragment thereof in a non-human vertebrate; and
(b) obtaining a serum from said non-human vertebrate.
55. The method according to claim 54, wherein said release factor 1 from Thermus thermophilus or said fragment thereof is purified.
56. The method according to claim 54 or 55, wherein said anti-release factor antibody as obtained in the eliciting-step (a) is purified.
57. The anti-release factor antibody according to any one of claims 45 to 52 or the method according to any one of claims 54 to 56, wherein said release factor 1 from Thermus thermophilus or said fragment thereof is a native release factor 1 from Thermus thermophilus or a fragment thereof.
58. A method for the production of alloproteins, comprising the step of translating a RNA into translation product in a cell-free translation system as defined in any one of claims 1 to 43.
59. The method according to claim 58, wherein said alloproteins comprising proteins selected from the group consisting of enzymes, hormones, cytokines, pheromones, growth factors, structural proteins, toxins, markers, reporters and the like.
60. The method according to claim 58 or 59, wherein said alloproteins comprising esterases.
61. The method according to any one of claims 58 to 60, wherein said alloproteins comprising the esterase 2 from Alicyclobacillus acidocaldarius.
62. The method according to any one of claims 58 to 61, wherein said alloproteins are proteins that covalently have attached puromycin or a derivative thereof; and/or have incorporated an (unnatural) amino acid delivered by aminoacyl suppressor tRNA.
63. The method according to any one of claims 58 to 62, wherein said alloproteins are proteins, that covalently have attached puromycin or a derivative thereof.
64. The method according to any one of claims 58 to 63, wherein said alloproteins are proteins that covalently have attached puromycin or a derivative thereof, wherein said puromycin or a derivative thereof is selected from the group consisting of:
(a) Puromycin;
(b) 5'-OH-CpPuromycin;
(c) 5'-OH-CpCpPuromycin;
(d) a puromycin derivative as defined in (a) to (c) having a residue covalently attached directly or via a linker to its 5'-position; (e) a puromycin derivative as defined in (a) to (d) having a residue covalently attached directly or via a linker to the element N4 of the cytosine-residue of an 5" attached cytidine-residue; and
(f) a puromycin derivative as defined in (a) to (e) having a residue covalently attached directly or via a linker to the element C5 of the cytosine-residue of an 5' attached cytidine-residue.
65. The method according to any one of claims 58 to 64, wherein the nonsense- codon suppressing agent comprises a puromycin derivative as defined in claim 64(a) to (d) having a residue covalently attached directly or via a linker to the element N4 of the cytosine-residue of an 5' attached cytidine-residue.
66. The method according to any one of claims 64 or 65, wherein said residue is selected from the group consisting of DNA, RNA, locked DNA, PNA, oligonucleotide-thiophosphates, substituted ribo-oligonucleotides, and proteins.
67. The method according to any one of claims 64 or 65, wherein said residue is selected from the group consisting of a fluorophore (like Cy3), biotin or an other affinity tag, a reactive group for affinity labelling of any other reporter group.
68. The method according to any one of claims 64 to 67, wherein said linker is an aliphatic amine derivative.
69. The method according to any one of claims 62 to 68, wherein the nonsense- codon suppressing agent is selected from the group consisting of:
(a) 5'-OH-GpCpPuromycin;
(b) 5'-OH-GpCpCpPuromycin;
(c) 5'-OH-GpApCpCpPuromycin;
(d) 5'-OH-GpCpApCpCpPuromycin;
(e) 5'-OH-GpCpCpApCpCpPuromycin;
Figure imgf000126_0001
Figure imgf000127_0001
Figure imgf000128_0001
Figure imgf000129_0001
70. The method according to any one of claims 58 to 69, wherein said alloproteins are proteins, that covalently have attached said puromycin or said derivative thereof at the C-terminus.
71. The method according to any one of claims 58 or 62, wherein said alloproteins are proteins having incorporated an (unnatural) amino acid delivered by aminoacyl suppressor tRNA.
72. The method according to any one of claims 58 to 62 and 71 , wherein said alloproteins are proteins having incorporated an (unnatural) amino acid delivered by aminoacyl suppressor tRNASer(CUA) .
73. A puromycin derivative having a residue covalently attached directly or via a linker to one of its elements selected from the group consisting of:
(a) the element N4 of the cytosine-residue of an 5' attached cytidine- residue; and
(b) the element C5 of the cytosine-residue of an 5' attached cytidine- residue.
74. The puromycin derivative according to claim 73, wherein said element is N4 of the cytosine-residue of a 5' attached cytidine-residue.
75. The puromycin derivative according to claim 73 or 74, wherein said residue is selected from the group consisting of DNA, RNA, locked DNA, PNA, oligonucleotide-thiophosphates, substituted ribo-oligonucleotides, and proteins.
76. The puromycin derivative according to claim 73 or 74, wherein said residue is selected from the group consisting of a fluorosphore (Cy3), biotin or another affinity tag, a reactive group for affinity labelling of any other reporter group.
77. The puromycin derivative according to any one of claims 73 to 76, wherein said linker is an aliphatic amine derivative.
78. The puromycin derivative according to any one of claims 73 to 77, wherein said puromycin derivative is selected from the group consisting of:
Figure imgf000132_0001
ı32
Figure imgf000133_0001
Figure imgf000134_0001
Figure imgf000135_0001
79. Use of a puromycin derivative having a residue covalently attached directly or via a linker to one of its elements selected from the group consisting of:
(a) the element N4 of the cytosine-residue of an 5' attached cytidine- residue; and
(b) the element C5 of the cytosine-residue of an 5' attached cytidine- residue; in a cell-free translation system as defined in any one of claims 1-43, in a method for the production of alloproteins according to any one of claims 58 to 72 or in a kit according to claim 44.
80. The use of a puromycin derivative according to claim 65, wherein said element is N4 of the cytosine-residue of an 5' attached cytidine-residue.
81. The use of a puromycin derivative according to claim 79 or 80, wherein said residue is selected from the group consisting of DNA, RNA, locked DNA, PNA, oligonucleotide-thiophosphates, substituted ribo-oligonucleotides, and proteins.
82. The use of a puromycin derivative according to claim 79 or 80, wherein said residue is selected from the group consisting of a fluorosphore (Cy3), biotin or another affinity tag, a reactive group for affinity labelling or any other reporter group.
83. The use of a puromycin derivative according to anyone of claim 79 to 82, wherein said linker is an aliphatic amine derivative.
84. The use of a puromycin derivative according to any one of claim 79 to 83, wherein said derivative is selected from the group consisting of:
Figure imgf000137_0001
Figure imgf000138_0001
Figure imgf000139_0001
Figure imgf000140_0001
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