# US 7,138,515 B2 · Translational activity-promoting higher-order structure > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 7,138,515 B2 - Document ID: US7138515B2 - Status: Granted - Kind: B2 - Application number: 10/088,750 - Filed: 2001-01-31 - Published: 2004-08-26 - Granted: 2006-11-21 - Earliest claimed: 2001-01-25 - Assignees: National Institute of Agrobiological Sciences - Inventors: Nobuhiko Nakashima; Yasushi Kanamori - Web page: https://patentgrep.com/patent/US7138515B2 ## CPC - C07K 14/005: from viruses - C12N 15/11: DNA or RNA fragments; Modified forms thereof (DNA or RNA not used in recombinant technology, C07H21/00); {Non-coding nucleic acids having a biological activity} - C12N 15/63: Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression - C12N 15/67: General methods for enhancing the expression - C12N 15/86: Viral vectors - C12N 2770/22022: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes - C12N 2770/22023: Virus like particles [VLP] - C12N 2840/203: having an IRES - C12N 2840/55: from bacteria - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) ## Abstract The present invention was conceived from the findings; that a specific higher-order structure can be formed in CrP-like viruses; that linkage of a gene reading frame downstream in the higher-order structure permits initiating the translation of a protein from various amino acids; and that a change in the combination of base pairs involved in the formation of the higher-order structure immediately upstream in the translation initiation site permits initiating the synthesis of any protein from any codon. ## Claims ### Claim 1 A method for synthesizing a heterologous protein or a heterologous polypeptide comprising the steps of providing a cell-free protein expressing system, providing a polynucleotide encoding the heterologous protein or heterologous polypeptide and a polynucleotide that promotes translation activity, wherein the polynucleotide that promotes translation activity has an RNA higher-order structure including PK (pseudoknot) I, II, and III structures and is selected from: 1) one of the base sequences of SEQ ID NOs: 1–7; 2) a base sequence of SEQ ID NO: 1, except that positions 187–188 of the base sequence of SEQ ID NO: 1 are cc instead of uu and positions 159–160 are gg instead of aa; and 3) a base sequence containing the base sequence of 1) or 2); and wherein the polynucleotide encoding the heterologous protein or heterologous polyptptide is immediately downstream from the PKI structure of the polynucleotide that promotes translation activity, translating the polynucleotide encoding the heterologous protein or heterologous polypeptide in the cell-free protein expressing system to form the heterologous protein or heterologous polypeptide, wherein the translating is initiated and/or promoted by the polynucleotide that promotes translation activity, and isolating the heterologous protein or heterologous polypeptide. ### Claim 2 (depends on 1) The method for synthesizing a heterologous protein or a heterologous polypeptide according to claim 1, wherein the synthesis is carried out without using AUG translation initiation codon. ### Claim 3 (depends on 1) The method for synthesizing a heterologous protein or heterologous polypeptide according to claim 1, wherein the RNA higher-order structure comprises a base sequence selected from one of the sequences of SEQ ID NOs:1–7. ### Claim 4 (depends on 3) The method for synthesizing a beterologous protein or a heterologous polypeptide according to claim 3 wherein the synthesis is carried out without using AUG translation initiation codon. ### Claim 5 (depends on 1) The method for synthesizing a heterologous protein or heterologous polypepfide according to claim 1, wherein the RNA higher-order structure comprises the base sequence of SEQ ID NO:1. ### Claim 6 (depends on 1) The method for synthesizing a heterologous protein or heterologous polypeptide according to claim 1, wherein the RNA higher-order structure comprises the base sequence of SEQ ID NO:1, except that positions 187–188 of the base sequence of SEQ ID NO:1 are cc instead of uu and positions 159–160 are gg instead of aa. ### Claim 7 (depends on 1) The method for synthesizing a heterologous protein or heterologous polypeptide according to claim 1 wherein the protein expressing system is a wheat germ extract. ### Claim 8 (depends on 7) The method for synthesizing a heterologous protein or a heterologous polypeptide according to claim 7, wherein the synthesis is carried out without using AUG translation initiation codon. ### Claim 9 A method for initiating synthesis of an arbitrary heterologous protein or heterologous polypeptide from an arbitrary codon which comprises the steps of providing a cell-free protein expressing system, providing a polynucleotide encoding the arbitrary heterologous protein or heterologous polypeptide and a polynucleotide that promotes translation activity, wherein the polynucleotide that promotes translation activity has PK (pseudoknot) I, II, and III structures in a RNA higher-order structure having a function for promoting a translation activity, wherein the polynucleotide encoding the heterologous protein or heterologous polyptptide is immediately downstream from the PKI structure of the polynucleotide that promotes translation activity and wherein the RNA higher-order structure has a base sequence selected from: 1) one of the base sequences of SEQ ID NOs: 1–7; 2) abase sequence of SEQ ID NO: 1, except that positions 187–188 of the base sequence of SEQ ID NO: 1 are cc instead of uu and positions 159–160 are gg instead of aa; and 3) a base sequence containing the base sequence of 1) or 2), translating the polynucleotide encoding the arbitrary heterologous protein or heterologous polypeptide from an arbitrary codon in the cell-free protein expressing system to form the heterologous protein or heterologous polypeptide, wherein the translating is initiated and/or promoted by the polynucleotide that promotes translation activity, and isolating the heterologous protein or heterologous polypeptide. ### Claim 10 (depends on 9) The method for initiating the synthesis according to claim 9, wherein the RNA higher-order structure comprises the base sequence of SEQ ID NO:1, except that positions 187–188 of the base sequence of SEQ ID NO:1 are cc instead of un and positions 159–160 are gg instead of aa.