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

Abstract

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 in vivo. 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 Nitrosomonas europaea 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.

Structures of Methanomethylophilus alvus Pyrrolysine tRNA-Synthetases Support the Need for De Novo Selections When Altering the Substrate Specificity

“…Moving forward, we found the Ma PylRS to be particularly amenable to structural characterization due to ease of expression and purification, unlike the Mb/Mm RS which is challenging to purify in its active state in large quantities and for which no structures of the full-length enzyme have been reported. − Also, while the functional, full-length Mm PylRS can only be concentrated to ∼4 mg/mL, the functional Ma RS protein is much more soluble (easily concentrated to over 30 mg/mL), and this feature was leveraged to improve yields for cell-free protein synthesis and facilitated our structural characterizations here as well as our in vitro kinetic characterizations described in the accompanying article. Thus, the tractability of the Ma PylRS/tRNA Pyl pair for in vitro studies should prove useful for expanding the utility of this GCE platform, particularly for amino acids that differ from a lysine-like scaffold.…”

Changes in Coding and Efficiency through Modular Modifications to a One Pot PURE System for In Vitro Transcription and Translation

“…alvus PylRS has been shown to be more tractable for engineering because it lacks an N-terminal domain ,− that leads to solubility issues for the M. barkeri PylRS. , Only the M. alvus PylRS system yielded the desired peptide product (Figure S3).…”

“…The M. barkeri PylRS was chosen because it had previously been shown to accept a wide variety of noncanonical amino acids, 22 while the M. alvus PylRS has been shown to be more tractable for engineering because it lacks an N-terminal domain 23,25−27 that leads to solubility issues for the M. barkeri PylRS. 22,28 Only the M. alvus PylRS system yielded the desired peptide product (Figure S3). However, there was noticeable misincorporation of tryptophan across from the opal codon, even with the addition of purified orthogonal suppressor tRNA UCA and ProgK (Figure S3).…”

Genomically integrated orthogonal translation inEscherichia coli, a new synthetic auxotrophic chassis with altered genetic code, genetic firewall, and enhanced protein expression

“…The efficiency of m -oNB-Dopa incorporation in the BNH25 strain indicates that the CAST tool is capable of elevating gene expression at the copy-number level in a synthetic bacterial chassis. This chassis robustly grows and can overexpress protein congeners with coding sequences containing up to 30 in-frame stop codons 32 .…”