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1. (EP2060584) Method for the production of proteins with chemically functionalized N-termini
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Claims

1. A method of producing an N-terminally modified polypeptide by translation from an RNA molecule in a host cell or in an in vitro translation system, wherein the RNA molecule encodes a polypeptide comprising an N-terminal methionine residue, wherein the translation occurs at conditions under which the N-terminal methionine residue is replaced by a non-genetically encoded amino acid residue and wherein thereby an N-terminal amino acid excision of the polypeptide is modified.
  2. The method of claim 1, wherein the host cell is a bacterial cell, particularly a gram-negative bacterial cell such as E. coli.
  3. The method of claim 1 or 2, wherein the host cell is a methionine-auxotrophic host cell.
  4. The method of any one of claims 1-3, wherein the polypeptide comprises a single methionine residue, which is the N-terminal methionine residue.
  5. The method of claims 1-3, wherein the polypeptide comprises an N-terminal methionine residue and at least one internal methionine residue.
  6. The method of claim 5, wherein only the N-terminal methionine residue is replaced by a non-genetically encoded amino acid.
  7. The method of claim 5, wherein the N-terminal methionine residue and at least one internal methionine residue are replaced by non-genetically encoded amino acid residues.
  8. The method of any one of claims 1-7, wherein the N-terminal amino acid excision is increased.
  9. The method of any one of claims 1-7, wherein the N-terminal amino acid excision is decreased.
  10. The method of any one of claims 1-9, wherein the penultimate amino acid residue after the N-terminal amino acid residue is an amino acid residue which affects N-terminal amino acid excision and/or polypeptide stability.
  11. The method of claim 10, wherein the penultimate amino acid residue is an amino acid residue which promotes N-terminal amino acid excision, particularly selected from the group consisting of Gly, Ala, Pro, Cys, Ser, Thr and Val.
  12. The method of claim 10, wherein the penultimate amino acid residue is an amino acid residue which increases polypeptide stability, particularly selected from the group consisting of Met, Gly, Ala, Ser, Thr and Val.
  13. The method of claim 10, wherein the penultimate amino acid residue is an amino acid residue which inhibits N-terminal amino acid excision, particularly selected from the group consisting of Arg, Lys, Phe, Leu and Ile.
  14. The method of claim 10, wherein the penultimate amino acid residue is an amino acid residue which decreases polypeptide stability, particularly selected from the group consisting of Arg, Lys, Asp, Leu, Ile and Phe.
  15. The method of any one of claims 1-14, wherein the ante-penultimate amino acid residue after the N-terminal amino acid residue is an amino acid residue which affects N-terminal amino acid excision and/or polypeptide stability.
  16. The method of claim 15, wherein the ante-penultimate amino acid residue is selected from Glu, Asp, Thr, Leu, Ile or Val.
  17. The method of any one of claims 1-16, wherein the non-genetically encoded amino acid residue comprises a functional chemical moiety.
  18. The method of claim 17, wherein the functional chemical moiety is selected from the group consisting of azide, alkene, alkyne, phosphine, halogen, or chalcogen, amine or ester moieties.
  19. The method of claim 18, wherein the functional chemical moiety is an aromatic and/or aliphatic azide or alkyne group.
  20. The method of any one of claims 1-19, wherein the non-genetically encoded amino acid residue is homopropargylglycine (Hpg) or azido homoalanine (Aha).
  21. The method of any one of claims 1-20, further comprising coupling a heterologous molecule to the functional chemical moiety of the non-genetically encoded amino acid residue.
  22. The method of claim 11, wherein the coupling comprises a [1,3]-dipolar cyclo-addition between an azide group and an alkyne group.
  23. The method of any one of claims 12 or 13, wherein the heterologous molecules are selected from the group consisting of reporter molecules, effector molecules, solid phases and nanomaterials.
  24. The method of any one of claims 1-23, wherein the polypeptide is selected from growth factors, viral antigens, annexins, antibodies, structure proteins, barstar, G-protein coupled receptors, green fluorescent protein and its spectral variants, human superoxide dismutase (hSOD1), spider silk and extracellular matrix proteins, proteases, kinases, phosphatases, hydroxylases, ligases or polymerases.
  25. A method of producing an N-terminally modified polypeptide by translation from an RNA molecule in a host cell or in an in vitro translation system, wherein the RNA molecule encodes an N-terminal polypeptide portion comprising an amino acid sequence which allows proteolytic cleavage and wherein the first amino acid residue behind the cleavage site is the amino acid residue to be modified, wherein the translation occurs at the conditions under which the amino acid residue to be modified is replaced by a non-genetically encoded amino acid residue, and wherein the polypeptide is subjected to a proteolytic cleavage at the cleavage site.
  26. The method of claim 25 wherein the amino acid residue to be modified is a methionine residue.
  27. The method of claim 25 or 26, wherein the amino acid residue to be modified only occurs once in the polypeptide after cleavage of the N-terminal polypeptide portion.
  28. A method of modifying efficacy of translation initiation, wherein an RNA molecule encoding a polypeptide comprising an N-terminal amino acid residue is translated in a host cell or in an in vitro translation system, wherein the translation occurs at conditions under which the N-terminal methionine residue is replaced by a non-genetically encoded amino acid residue and wherein thereby the efficacy of translation initiation is modified, e.g. decreased or increased.
  29. An N-terminally modified polypeptide comprising at its N-terminus a non-genetically encoded amino acid residue, obtainable by the method of any one of claims 1-28.
  30. The polypeptide of claim 29, wherein the non-genetically encoded amino acid residue comprises a functional chemical moiety.
  31. The polypeptide of claim 30, wherein the functional chemical moiety is coupled to a heterologous molecule.