Transforming a Pair of Orthogonal tRNA-aminoacyl-tRNA Synthetase from Archaea to Function in Mammalian Cells

Abstract

A previously engineered Methanocaldococcus jannaschii –tyrosyl-tRNA synthetase pair orthogonal to Escherichia coli was modified to become orthogonal in mammalian cells. The resulting -tyrosyl-tRNA synthetase pair was able to suppress an amber codon in the green fluorescent protein, GFP, and in a foldon protein in mammalian cells. The methodology reported here will allow rapid transformation of the much larger collection of existing tyrosyl-tRNA synthetases that were already evolved for the incorporation of an array of over 50 unnatural amino acids into proteins in Escherichia coli into proteins in mammalian cells. Thus we will be able to introduce a large array of possibilities for protein modifications in mammalian cells.

Crucial Optimization of Translational Components towards Efficient Incorporation of Unnatural Amino Acids into Proteins in Mammalian Cells

“…Six tandem repeats of the resulting genes were cloned into plasmid pZeoSV2 (+) to create 6x_wt-tRNA CUA ( Figure 1 ) [11]. However, this approach afforded low expression of the tRNA in mammalian cells due to the differences in the manner in which archaeal and eukaryotic cells initiate transcription of their tRNA genes and subsequently process the transcript [12].…”

“…To the best of our knowledge, only a few unnatural aaRSs are available for incorporating UAAs in eukaryotic cells [9], as compared to the several dozens that can be used in prokaryotic cells. Evidence suggests that those domains on the TyrRS responsible for recognizing the amino acid and tRNA targets can be modified independently of one another [10], [11]. The existing set of unnatural aaRS derived from M. jannaschii TyrRS can thus be used in a eukaryotic system if they are manipulated into ignoring endogenous tRNAs.…”

“…We have previously shown that M. jannaschii TyrRS can be made orthogonal in mammalian cells by using peptide transplantation involving the connective polypeptide 1 (CP1) domain of E. coli TyrRS [11]. M. jannaschii that is not orthogonal in mammalian cells can be made orthogonal by switching C1:G72 to G1:C72 thus generating 1bp-tRNA CUA [11].…”

“…M. jannaschii that is not orthogonal in mammalian cells can be made orthogonal by switching C1:G72 to G1:C72 thus generating 1bp-tRNA CUA [11]. The newly designed mutant TyrRS can then be paired with the engineered orthogonal amber codon-suppressing tRNA to form an orthogonal pair in mammalian cells capable of introducing a tyrosine to a protein in response to amber codon TAG.…”

“…The newly designed mutant TyrRS can then be paired with the engineered orthogonal amber codon-suppressing tRNA to form an orthogonal pair in mammalian cells capable of introducing a tyrosine to a protein in response to amber codon TAG. We reasoned that a similar CP1 transplantation could also be applied to an evolved M. jannaschii TyrRS (aaRS) specific to an unnatural amino acid in E. coli allowing it to charge 1bp-tRNA CUA with the same unnatural amino acid in mammalian cells [11]. However, low suppression efficiency limited our ability to manipulate an unnatural aaRS in this manner.…”