PURPOSE: To obtain the subject substance into which a non-natural amino acid is inserted at an arbitrary position required by an user, by controlling the insertion position of the non-natural amino acid for each codon group by a specific method using a cell-free protein synthesis system.
CONSTITUTION: A non-natural amino acid-inserted protein is synthesized in a cell-free protein synthesis system using (A) the combination product of the non-natural amino acid with an isoaccepting t-RNA corresponding to a specific codon and (B) amino acids constituting the protein as subtrates to provide the objective substance in which the insertion position of the non-natural amino acid is controlled for each codon group. A continuous cell-free polypeptide synthesis system is preferable as the above-mentioned cell-free protein synthesis system. The synthesis system is preferably a system which is obtained e.g. by a method in which an antibody against an aminoacyl t-RNA synthesis enzyme is added and in which the activity of the aminoacyl t-RNA synthesis enzyme corresponding to the used isoaccepting t-RNA is low.
KIKAWA TAKANORI
TEJIMA MUNEHIRO
