CELL-FREE SYNTHETIC INCORPORATION OF NON-NATURAL AMINO ACIDS INTO PROTEINS

摘要

The present disclosure provides methods for incorporating at least one non-natural amino acid into a polypeptide using a cell-free protein synthesis which includes a cell-free extract and is deficient in endogenous tRNA. The methods include providing the synthesis system with at least one non-natural amino acid, at least one orthogonal tRNA and one orthogonal aminoacyl-tRNA synthetase which aminoacylates the corresponding orthogonal tRNA with the non-natural amino acid. The methods may also include removing the endogenous tRNA in the cell-free protein synthesis system by treating the synthesis system with a ribonuclease to degrade the endogenous tRNA and providing the synthesis system with a minimal set of tRNAs for one or more natural amino acids and the corresponding amino-acyl tRNA synthetases, such that the lack of some tRNA will enable unique codons to encode for non-natural amino acid incorporation with no or a minimal amount of completion from the tRNA present.

主权项

1. A method for incorporating at least one non-natural amino acid into a polypeptide using a cell-free protein synthesis system, wherein the cell-free protein synthesis system comprises a cell-free extract and is deficient in endogenous tRNA, the method comprising:
providing the synthesis system with the at least one non-natural amino acid, at least one orthogonal aminoacyl-tRNA synthetase and an orthogonal tRNA corresponding to the at least one non-natural amino acid, wherein the orthogonal aminoacyl-tRNA synthetase aminoacylates the corresponding orthogonal tRNA with the non-natural amino acid.