Efficient Unnatural Protein Production by Pyrrolysyl-tRNA Synthetase With Genetically Fused Solubility Tags

Introducing non-canonical amino acids (ncAAs) by engineered orthogonal pairs of aminoacyl-tRNA synthetases and tRNAs has proven to be a highly useful tool for the expansion of the genetic code. Pyrrolysyl-tRNA synthetase (PylRS) from methanogenic archaeal and bacterial species is particularly attractive due to its natural orthogonal reactivity in bacterial and eukaryotic cells. However, the scope of such a reprogrammed translation is often limited, due to low yields of chemically modified target protein. This can be the result of substrate specificity engineering, which decreases the aminoacyl-tRNA synthetase stability and reduces the abundance of active enzyme. We show that the solubility and folding of these engineered enzymes can become a bottleneck for the production of ncAA-containing proteins . Solubility tags derived from various species provide a strategy to remedy this issue. We find the N-terminal fusion of the small metal binding protein from to the PylRS sequence to improve enzyme solubility and to boost orthogonal translation efficiency. Our strategy enhances the production of site-specifically labelled proteins with a variety of engineered PylRS variants by 200-540%, and further allows triple labeling. Even the wild-type enzyme gains up to 245% efficiency for established ncAA substrates.