New England Biolabs, N6845S, E. coli tRNA

Related Categories PURExpress® In Vitro Protein Synthesis,, Cell-Free Protein Expression,, Protein Purification, Applications PURExpress,, Protein Expression in, E. coli FAQ Q: Can I use E. coli tRNA (NEB #N6845) with yeast, insect, mammalian or other eukaryotic translation systems? A: Although translation mechanisms are very similar in prokaryotic and eukaryotic systems, the initiation steps show important differences: for instance, in eukaryotic systems, methionyl-tRNA (Met-tRNAi) is not formylated and it interacts (initiates) with the ribosome as a complex with eIF2 and GTP. Therefore, a mix of prokaryotic tRNA will lack all necessary attributes to sustain effective protein synthesis in the presence of eukaryotic ribosomes. Q: How is E. coli tRNA (NEB #N6845) purified? A: E. coli tRNA N6845 is extracted from E. coli cells. The transfer RNA is purified by a series of chromatography separations, directly from a cell lysate and avoiding any precipitation step that involves organic solvents. The final solution is provided in 10 mM Tris-HCl buffer (pH 7.5), and it is free of organic contaminants. Q: I want to supplement with an unnatural amino acid and would like to know the individual tRNA composition. A: The final concentration of all the tRNAs in this product is 35 mg/mL. That said, the tRNAs are purified as a total pool from E. coli, and we assume that they are present in the ratio in which they exist in vivo. Q: If my template is not optimized for E. coli, do I have to supplement E. coli tRNA (NEB #N6845) with rare codon tRNAs? A: Frequent truncations are a sign that translation may stall at rare codon positions. Rather than supplementing rare tRNAs (which are challenging to purify), a better strategy is to codon-optimize your template. Although the mere presence of a rare codon could affect translation efficiency, this is not the only cause of poor translation or truncations. Other factors can have a much larger effect: how many consecutive rare codons are present, target sequence (5’ and 3’ UTR regions, N- or C-terminal tags), template concentration, and incubation conditions. Q: What applications are compatible with E. coli tRNA (NEB #N6845)? A: This tRNA is ready to use for in vitro protein translation in E. coli systems, ribosome display, mRNA display, and other molecular biology applications.