Methods for ribosomal synthesis of polypeptides containing unnatural N-terminal groups and applications thereof
AssigneeThe University of Tokyo
InventorsHiroaki Suga, Hiroshi Murakami, Yuki Goto, Atsushi Ohta
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Abstract
The present invention aims to synthesize a polypeptide having an unnatural structure at the N-terminus via a biosynthetic process by translation of amino acid sequence information encoded by a nucleic acid. A polypeptide having any amino acid at the N-terminus is synthesized by using an ARS ribozyme that catalyzes the acylation of tRNA with any amino acid to attach any amino acid to an initiator tRNA, thereby initiating a translation with the initiator tRNA.
Claims17
A process for translationally synthesizing a polypeptide having a desired N-terminal structure, comprising the steps of:
- (a) providing a ribozyme capable of catalyzing the acylation reaction of tRNA;
- (b) providing an amino acid substrate having a desired structure for use as a substrate for the acylation reaction by the ribozyme;
- (c) performing an acylation reaction of an initiator tRNA with the amino acid substrate in (b) above using the ribozyme in (a) above to give an initiator tRNA aminoacylated with the amino acid having a desired structure;
- (d) adding the aminoacylated initiator tRNA obtained in (c) above to a cell-free translation system lacking methionine or methionyl-tRNA synthetase (MetRS) to initiate a translation with the amino acid having a desired structure, thereby giving a polypeptide having a desired N-terminal structure,
- wherein said amino acid that aminoacylates the initator tRNA in step (c) is a common amino acid other than methionine and is selected from the group consisting of alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), proline (Pro), tryptophan (Trp), phenylalanine (Phe), glycine (Gly), serine (Ser), threonine (Thr), tyrosine (Tyr), cysteine (Cys), glutamine (Gln), asparagine (Asn), lysine (Lys), arginine (Arg), histidine (His), aspartate (Asp), and glutamate (Glu), or said amino acid is an unusual amino acid.
A process for translationally synthesizing a polypeptide having a desired N-terminal structure, comprising the steps of:
- (a) providing as ribozyme capable of catalyzing the acylation reaction of tRNA;
- (b) providing an amino acid substrate having a desired structure for use as a substrate for the acylation reaction by the ribozyme;
- (c) performing an acylation reaction of an initiator tRNA that has an anticodon other than CAU with the amino acid substrate in (b) above using the ribozyine in (a) above to give an initiator tRNA that has an anticodon other than CAU aminoacylated with the amino acid having a desired structure;
- (d) adding the aminoacylated initiator tRNA obtained in (c) above to a cell-free translation system to initiate a translation with the amino acid having a desired structure, thereby giving a polypeptide having a desired N-ternainal structure,
- wherein said amino acid that aminoacylates the initiator tRNA in step (c) is a common amino acid other than methionine and is selected from the group consisting of alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), proline (Pro), tryptophan (Trp), phenylalanine (Phe), glycine (Gly), serine (Ser), threonine (Thr), tyrosine (Tyr), cysteine (Cys), glutamine (Gln), asparagine (Asn), lysine (Lys), arginine (Arg), histidine (His), aspartate (Asp), and glutamate (Glu), or said amino acid is an unusual amino acid.
Dependencies are inferred from the claim text.