Process for production of non-natural protein having ester bond therein

A method of producing a cell or a cell extract having activity of incorporating an alpha-hydroxy acid into a polypeptide to provide a polypeptide having an ester bond in the main chain, comprising expressing

• (a1) a pyrrolysyl-tRNA synthetase derived from an archaebacterium that is capable of activating an α-hydroxy acid that is N-substituted 6-amino-2-hydroxyhexanoic acid;
• or
• (a2) a pyrrolysyl-tRNA synthetase having the amino acid sequence of SEQ ID NO:
• 2 and having at least one amino acid substitution of:
• from tyrosine to alanine at position 306,
• from leucine to alanine at position 309,
• from cysteine to valine at position 348, and
• from tyrosine to phenylalanine at position 384,
• and that is capable of activating an α-hydroxy acid that is N-substituted 6-amino-2-hydroxyhexanoic acid;
• or
• (a3) pyrrolysyl-tRNA synthetase having the amino acid sequence of SEQ ID NO:
• 2 and having the five amino acid substitutions from alanine to phenylalanine at position 302, tyrosine to alanine at position 306, from asparagine to serine at position 346, from cysteine to isoleucine at position 348, and from tyrosine to phenylalanine at position 384, and that is capable of activating an α-hydroxy acid that is an optionally substituted 3-phenyllactic acid;
• (b) a suppressor tRNA capable of binding to the α-hydroxy acid in the presence of said pyrrolysyl-tRNA, synthetase;
• and
• (c) a gene encoding a desired protein that has a nonsense mutation or frameshift mutation at a desired site;
• in a cell or cell extract solution in the presence of said α-hydroxy acid.