Artificial translation/synthesis system

Patent metadata

Field	Value
Publication number	US-9701993-B2
Application number	US-201113816911-A
Country	US
Kind code	B2
Filing date	Aug 26, 2011
Priority date	Aug 27, 2010
Publication date	Jul 11, 2017
Grant date	Jul 11, 2017

Abstract

A new artificial translation-synthesis system of adding tRNAs binding special amino acids to the in vitro translation system and synthesizing peptides with special amino acids incorporated thereto according to a dual genetic code table and an artificial codon box division.

First claim

The invention claimed is:

1. A method for synthesizing peptides using dual sense codons and artificial division of codon boxes, comprising:
i) combining at least the following in an in vitro translation system:
(a) mRNAs;
(b) initiator tRNA(s) each charged with a (b) artificial amino acid;
(c) elongator tRNA(s) each charged with a (c) natural amino acid, wherein each of the (c) tRNA(s) is independently complementary to first codon(s) of the mRNA of its (c) natural amino acid in the genetic code;
(d) elongator tRNA(s) each charged with a (d) artificial amino acid, wherein at least one of the (d) tRNA(s) is independently complementary to second codon(s) of the mRNA of an (c) natural amino acid in the genetic code;
(e) optionally, initiator tRNA(s) and/or elongator tRNA(s) each charged with a natural or an artificial amino acid,
wherein a codon for each (b) initiator tRNA is the same as a codon for a (c) elongator tRNA or a (d) elongator tRNA; and

ii) synthesizing at least one peptide comprising a (b) artificial amino acid, a (c) natural amino and a (d) artificial amino acid using the mRNAs and tRNA(s) in the in vitro translation system,
wherein a codon for a (b) initiator tRNA of the (b) artificial amino acid is the same as a codon for a (c) elongator tRNA of the (c) natural amino acid or a codon for a (d) elongator tRNA of the (d) artificial amino acid, and
wherein the (b) artificial amino acid and the (d) artificial amino acid in the at least one peptide are different if the codon for the (b) initiator tRNA is the same as the codon for the (d) elongator tRNA.

2. The method according to claim 1 , wherein the (b) initiator tRNA(s) each charged with a (b) artificial amino acid and (d) elongator tRNA(s) each charged with a (d) artificial amino acid are prepared by an in vitro aminoacylation of tRNAs formed by in vitro transcription.

3. The method according to claim 1 , wherein the
(c) natural amino acid is one or more selected from Leu, Val, Ser, Pro, Thr, Ala, Arg, and Gly.

4. The method according to claim 3 , wherein a codon for a (e) elongator tRNA charged with an (e) artificial amino acid may be a termination codon selected from UAG, UAA and UGA.

5. The method according to claim 1 or 2 , wherein the translation system comprises in vitro transcribed tRNAs that are capable of being charged with the natural amino acids by the catalysis of endogenous ARSs.

6. The method according to claim 1 or 2 , wherein the codon assigning the translation initiation is AUG.

7. The method according to claim 1 or 2 , wherein codon(s) for (b) initiator tRNA(s) are selected from AUG, UGG, AUC, ACC, UCG, AAC, GCC, GGC, CCG, CGG, GGG, and AUA.

8. The method according to claim 6 , wherein AUG is a codon for a (d) elongator tRNA charged with an (d) artificial amino acid.

9. The method according to claim 7 , wherein codon(s) for (b) initiator tRNA(s) are selected from AUG, UGG, AUC, ACC, UCG and ACC.

10. A kit for an in vitro translation system that expresses a nonstandard peptide using dual sense codons and artificial division of codon boxes, comprising at least:
(a) a ribosome
(b) initiator tRNA(s) each charged with a (b) artificial amino acid;
(c) elongator tRNA(s) each charged with a (c) natural amino acid, wherein each of the (c) tRNA(s) is independently complementary to first codon(s) of the mRNA of its (c) natural amino acid in the genetic code;
(d) elongator tRNA(s) each charged with a (d) artificial amino acid, wherein at least one of the (d) tRNA(s) is independently complementary to second codon(s) of the mRNA of an (c) natural amino acid in the genetic code,
(e) optionally, initiator tRNA(s) and/or elongator tRNA(s) each charged with a natural or an artificial amino acid,
wherein a codon for each (b) initiator tRNA is the same as a codon for a (c) elongator tRNA or a (d) elongator tRNA.

11. The method according to claim 1 or 2 , wherein a codon for a (b) initiator tRNA can be selected from any triplet sequence.

Assignees

• Suga Hiroaki
• Murakami Hiroshi
• Goto Yuki
• Univ Tokyo

Inventors

• Suga Hiroaki
• Murakami Hiroshi
• Goto Yuki

Classifications

• C12P21/00 Primary

  Preparation of peptides or proteins (single cell protein C12N1/00) · CPC title

• C12P21/02

  having a known sequence of two or more amino acids, e.g. glutathione · CPC title

• C12N15/67 Primary

  General methods for enhancing the expression · CPC title