Separation of the BPY-labeled short peptides produced by a variety of systems. (A) Comparison of electrophoretic mobilities of MF2 (3, 4) and MF4 (5, 6) peptides obtained in a course of coupled transcription–translation process (3, 5) and those of chemically synthesized peptides (4, 6). The components added to the translation reaction or loaded to the gel (in the case of chemically synthesized peptides) are designated above the lanes. BPY is marked with a green oval—BODIPY label; black circle—methionine; gray circles—phenylalanine residues; blue oval—tRNAfMet. Lanes 1 and 2 correspond to the BPY-M-tRNAfMet and BPY-M, respectively. (B) Electrophoretic separation of MF and MF6 peptides produced in a translation system assembled from the purified components with premade mRNA. Designations are the same as for panel A. (C) Separation of dipeptides (BPY-M[14C]F, fM[14C]F), synthesized by in vitro translation reconstituted from the purified components, by HPLC in convex gradient of acetonitrile as described in Materials and Methods. BPY-labeled MF dipeptide is eluted four fractions later than nonlabeled MF dipeptide (depicted by arrow). (D) Comparison of the suitability of the BPY-M-tRNAfMet samples produced with a “homemade” sulfosuccinimide ether of BPY (lanes 1–3) or commercial sulfosuccinimide ether of BPY (lanes 4–6) for in vitro translation, reconstituted from the purified components, of MF-coding mRNA. Designations are the same as for panel A.
