The use of pLysB19, a new plasmid, for in vitro transcription of milligram quantities of human lysyl tRNA and purification by urea denaturing PAGE

A method for production of milligrams of hKtRNA, using new template plasmid pLysB19, by in vitro transcription is presented and compared to methods in use elsewhere. The in vitro transcription yield was found to be almost exclusively dependent on Mg²⁺ and template concentration. Currently, production of milligrams of hKtRNA is possible but only by use of higher template concentrations than reported elsewhere (7μg vs. 1μg per 10μL reaction). The typical yield of hKtRNA is ≥10μg per 10μl of transcription reaction mixture, of which approximately 6.5μg is recovered after gel purification. Up to 75% of the in vitro transcribed hKtRNA can be aminoacylated by human lysyl tRNA synthetase on acidic urea denaturing PAGE. The requirement of higher template concentrations than reported elsewhere to achieve similar yields is probably due to reduced T7RNAP activity; but the exact cause cannot be stated with absolute certainty due to multiple variations between the in vitro transcription reaction described here and reactions previously described elsewhere. However, recent results by others in this group show lower template concentrations used in the system described here can produce higher yields than shown here when different batches of T7RNAP are used. This is very supportive of conclusions here that reduced T7RNAP activity was the basis for the requirement of higher template concentrations than reported elsewhere.

Large-scale production of new template plasmid, pLysB19, is achieved by regeneration and re-use of silica membrane and ion exchange columns provided in commercial kits. Design and construction of pLysB19, done elsewhere, is also described. The transcribed hKtRNA is purified to ~95% on denaturing urea PAGE. The cause of the ~5% degradation products is explored through incubations of hKtRNA in high and low magnesium, as well as high and low pH buffers to determine whether these factors contribute to transcript degradation. The transcript degradation product pattern did not appear to be affected at Mg²⁺ concentrations as high as 207mM, or during incubations in pH 6 gel elution buffer. These results suggest that degradation under the purification conditions (37 °C for 12 hours at pH8) are not due to the reported instability of RNA in the presence of Mg²⁺ at elevated temperature or pH.

A method is also adapted for full resolution of the aminoacylated hKtRNA band from non-aminoacylated hKtRNA on longer acidic urea denaturing PAGE than previously described. A method for comparing the activities of hKRS using a 3H lysine charging assay is also presented and preliminary results are shown. In summary, the results and methods described here may serve as a guide for future experiments which require preparation of milligrams of in vitro transcribed hKtRNA, or the resolution of aminoacylated hKtRNA from non-aminoacylated hKtRNA on acidic urea PAGE.