In vitro reconstitution of the Escherichia coli 70S ribosome with a full set of recombinant ribosomal proteins

Abstract

Many studies of the reconstitution of the Escherichia coli small ribosomal subunit from its individual molecular parts have been reported, but contrastingly, similar studies of the large ribosomal subunit have not been well performed to date. Here, we describe protocols for preparing the 33 ribosomal proteins of the E. coli 50S subunit and demonstrate successful reconstitution of a functionally active 50S particle that can perform protein synthesis in vitro. We also successfully reconstituted both ribosomal subunits (30S and 50S) and 70S ribosomes using a full set of recombinant ribosomal proteins by integrating our developed method with the previously developed fully recombinant-based integrated synthesis, assembly and translation. The approach described here makes a major contribution to the field of ribosome engineering and could be fundamental to the future studies of ribosome assembly processes.

Keywords: PURE system; cell-free protein synthesis; protein translation; ribosomal protein; ribosome assembly.

Graphical Abstract

Fig. 1

Purified recombinant ribosomal proteins for the 50S subunit. The uL1, uL2-bL27 and bL28-bL36 were analysed with 15% and 17% SDS-PAGE and 16.5% Urea-Tris/Tricine-SDS-PAGE, respectively. The gels were stained with Coomassie Brilliant Blue G-250.

Fig. 2

SDGC analysis of reconstituted particles. Native 50S subunits, reconstituted subunits with TP50 (r50S-TP50 and r50S-TP50-Heat(−)) and 33 recombinant ribosomal proteins (r50S-33P and r50S-33P-Heat(−)) and native 5S and 23S rRNA mixture were analysed. The r50S-TP50 and r50S-33P were prepared by the normal procedure and r50S-TP50-Heat(−) and r50S-33P-Heat(−) were prepared without heat activation, in which all steps were performed on ice.

Fig. 3

LC–MS analysis of reconstituted 50S subunits. The ribosomal proteins in fractions separated via SDGC were analysed with LC–MS in a semi-quantitative manner. Recombinant 50S subunits were generated using 33P or TP50. The ratio of the signal intensity of each peptide peak relative to that of the corresponding peptide in the native 50S subunit was calculated. The ratios were then normalized to the average uL4 peptide signal. Each dot represents the signal intensity ratio of an individual peptide listed in Supplementary Data 7.

Fig. 4

Protein synthesis activities of reconstituted 50S subunits. Protein synthesis activities of native 50S subunits (n50S), reconstituted subunits with TP50 (r50S (TP50)) and 33 recombinant ribosomal proteins (r50S (33P)), reconstituted subunits only with 5S and 23S rRNA mixture (r50S (−proteins)) and only with 33 proteins (r50S (33P, -rRNAs)) were measured by performing HiBiT-tag peptide synthesis (a) and sfGFP synthesis (b) in the PURE system. End-point luminescence intensity exhibited by the interaction between LgBiT protein and synthesized HiBit-tag (a) and time-lapse changes of fluorescence intensity of synthesized sfGFP (b) are shown.

Fig. 5

Integration with the R-iSAT. Protein synthesis activities of reconstituted ribosomes using native 50S subunits (n50S), reconstituted subunits with TP50 (r50S (TP50)) and 33 recombinant ribosomal proteins (r50S (33P)), reconstituted subunits only with 5S and 23S rRNA mixture (r50S (−proteins)) and only with 33 proteins (r50S (33P, -rRNAs)) were measured by performing sfGFP synthesis in the R-iSAT. Time-lapse changes of fluorescence intensity of synthesized sfGFP (a) and fluorescent images of sfGFP in 15% SDS-PAGE (b) are shown.