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

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.

Automated summary

This study successfully reconstituted the 50S and 30S subunits of Escherichia coli ribosomes, as well as the 70S ribosomes, and demonstrated their ability to perform functional protein synthesis in vitro. This is a significant contribution to the field of ribosome engineering and the study of ribosome assembly processes.