Decoupling Protein Production from Cell Growth Enhances the Site-Specific Incorporation of Noncanonical Amino Acids in <i>E. coli</i>

Abstract

The site-specific incorporation of noncanonical amino acids (ncAAs) into proteins by amber stop codon suppression has become a routine method in academic laboratories. This approach requires an amber suppressor tRNA_CUA to read the amber codon and an aminoacyl-tRNA synthetase to charge the tRNA_CUA with the ncAA. However, a major drawback is the low yield of the mutant protein in comparison to the wild type. This effect primarily results from the competition of release factor 1 with the charged suppressor tRNA_CUA for the amber codon at the A-site of the ribosome. A number of laboratories have attempted to improve the incorporation efficiency of ncAAs with moderate results. We aimed at increasing the efficiency to produce high yields of ncAA-functionalized proteins in a scalable setting for industrial application. To do this, we inserted an ncAA into the enhanced green fluorescent protein and an antibody mimetic molecule using an industrial <i>E. coli</i> strain, which produces recombinant proteins independent of cell growth. The controlled decoupling of recombinant protein production from cell growth considerably increased the incorporation of the ncAA, producing substantially higher protein yields <i>versus</i> the reference <i>E. coli</i> strain BL21(DE3). The target proteins were expressed at high levels, and the ncAA was efficiently incorporated with excellent fidelity while the protein function was preserved.