Transfer RNAs (tRNA) are highly structured, non-coding RNA molecules that facilitate protein translation by delivering cognate amino acids to the ribosome. E. coli tRNA is a mix of tRNAs isolated from E. coli and purified by chromatography to remove contaminating proteins, including nucleases, as well as other nucleic acids. This tRNA formulation sustains translation in E. coli-based in vitro protein synthesis reactions (also referred to as cell-free protein synthesis or CFPS). It is also suited for molecular biology applications that require highly pure and active prokaryotic tRNA.
Source:
Extracted from E. coli cells and purified by a chromatographic process that avoids precipitation steps and obviates the use of harmful organic solvents, rendering it free of organic solvent contaminants and eliminating disposal of hazardous waste for reduced environmental impact.
Figure 1: E. coli tRNA isolated by chromatography is purer than tRNA extracted with organic solvents
Size comparison of E. coli tRNA purified by chromatography (NEB #N6845) and other E. coli tRNA obtained by organic solvent extraction. Samples of tRNA (35 ng) were separated by TBE-Urea PAGE (15% gel). In this system, the total tRNA population appears as a series of discrete bands, ranging from approximately 70 to 150 nt (indicated by a box). In contrast, in another tRNA sample purified by conventional organic solvent extraction, impurities persist and appear as smeared bands of higher and lower molecular weights. Figure 2: E. coli tRNA sustains synthesis of highly active protein in PURExpress® reactions
PURExpress cell-free protein synthesis reactions using E. coli tRNA yield functional protein. Luciferase activity and fluorescence signal were detected in crude cell-free protein synthesis samples. Reactions were performed using the PURExpress Δ (aa, tRNA) Kit (NEB #E6840) using 87.5 µg of E. coli tRNA (NEB #N6845) and 250 ng plasmid either encoding Firefly Luciferase or super-thermal Green Fluorescent Protein, in a total volume of 25 µl, incubated at 37°C for 2 hours. Figure 3: E. coli tRNA can support the synthesis of targets of different sizes and lengths
SDS-PAGE of crude cell-free protein synthesis reactions performed with the PURExpress Δ (aa, tRNA) Kit (NEB #E6840) using 87.5 µg of E. coli tRNA (NEB #N6845). Reaction conditions: template DNA; 250 ng plasmid encoding Dihydrofolate reductase (DHFR), super-thermal Green Fluorescent Protein (stGFP), Firefly Luciferase (FLuc) or β-Galactosidase (βGal) in a total volume of 25 µl, incubated at 37°C for 2 hours. A total of 2.5 µl of crude PURExpress reaction was loaded in each lane, with 10 µl of protein ladder marker L = (NEB #P7719). The red dots indicate the target protein synthesized in each reaction.
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E. coli tRNA is provided in 10mM Tris HCl at a concentration of 35 mg/mL. This tRNA is ready to use for in vitro protein translation / CFPS, ribosome display, mRNA display, and other molecular biology applications. For in vitro protein synthesis reactions, start by adding 1/10th the total volume of the reaction (e.g. add 2.5 µL of E. coli tRNA to a 25 µL total reaction). Titrate as needed to determine optimal conditions.
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