Dual amino acid-selective and site-directed stable-isotope labeling of the human c-Ha-Ras protein by cell-free synthesis

We developed two methods for stable-isotope labeling of proteins by cell-free synthesis. Firstly, we applied cell-free synthesis to the dual amino acid-selective C-13-N-15 labeling method, originally developed for in vivo systems by Kainosho and co-workers. For this purpose, we took one of the advantages of a cell-free protein synthesis system; the amino acid-selective stable-isotope labeling is free of the isotope scrambling problem. The targets of selective observation were Thr(35) and Ser(39) in the 'effector region' (residues 32-40) of the Ras protein complexed with the Ras-binding domain of c-Raf-1 (Raf RED) (the total molecular mass is about 30 kDa). Using a 15-mL Escherichia coli cell-free system, which was optimized to produce about 0.4 mg of Ras protein per 1-mL reaction, with 2 mg each of DL-[C-13']proline and L-[N-15]threonine, we obtained about 6 mg of Ras protein. As the Pro-Thr sequence is unique in the Ras protein, the Thr(35) cross peak of the Ras Raf RED complex was unambiguously identified by the 2D H-1-N-15 HNCO experiment. The Ser(39) cross peak was similarly identified with the [C-13']Aspi/[N-15]Ser-selectively labeled Ras protein. There were no isotope scrambling problems in this study. Secondly, we have established a method for producing a milligram quantity of site-specifically stable-isotope labeled protein by a cell-free system involving amber suppression. The E. coli amber suppressor tRNA(CUA)(Tyr) (25 mg) was prepared by in vitro transcription with T7 RNA polymerase. We aminoacylated the tRNA(CUA)(Tyr) transcript with purified E. coli tyrosyl-tRNA synthetase, using 2 mg of L-[N-15]tyrosine. In the gene encoding the Ras protein, the codon for Tyr(32) was changed to an amber codon (TAG). This template DNA and the [N-15]Tyr-tRNA(CUA)(Tyr) were reacted for 30 min in 30 mt of E. coli cell-free system. The subsequent purification yielded 2.2 mg of [N-15]Tyr(32)-Ras protein. In the H-1-N-15 HSQC spectrum of the labeled Ras protein, only one cross peak was observed, which was unambiguously assigned to Tyr(32).