Application of high-performance liquid chromatography to the reconstitution of ribosomal subunits

We are currently utilizing reversed-phase high-performance liquid chromatography (RP-HPLC) in reconstitution experiments designed to study the structure and function of Escherichia coli ribosomes. The applications of RP-HPLC in these experiments include: (a) preparation of individual proteins or groups of proteins on a milligram scale for reconstitution pools, (b) analysis of the protein stoichiometry of reconstituted subunits, (c) determination of the extent and specificity of modification of proteins extracted from ribosomal subunits which have been subjected to chemical modification, and (d) resolution of modified forms of proteins S14 and L23 from the corresponding unmodified proteins. Proteins prepared by RP-HPLC from 30S and 50S ribosomal subunits were found to reconstitute into 30S and 50S subunits respectively, as well as into slower sedimenting particles. The reconstituted subunits contain a full complement of proteins and are active in ribosomal function assays, whereas the slower sedimenting particles lack several proteins and have little or no activity.