Site-specific incorporation of unnatural amino acids into proteins by cell-free protein synthesis

Authors

Kiyoshi Ozawa, University of WollongongFollow
Choy Theng Loh, Australian National University

RIS ID

86411

Publication Details

Ozawa, K. & Loh, C. Theng. (2014). Site-specific incorporation of unnatural amino acids into proteins by cell-free protein synthesis. Methods in Molecular Biology, 1118 189-203.

Abstract

Cell-free protein synthesis (CFPS) offers a fast and inexpensive means to incorporate unnatural amino acids (UAAs) site specifically into proteins. This enables engineering of proteins and allows production of protein-based probes for analysis of their interactions with other molecules. Using dialysis Escherichia coli CFPS system in combination with aminoacyl-tRNA synthetase and suppressor tRNA evolved from Methanocaldococcus jannaschii high expression yield of proteins with site specifically incorporated UAAs can be achieved. Typically the target protein can be prepared at concentrations of about 1 mg/mL, which is generally sufficient for subsequent applications.