Open AccessIn vitroreconstitution of theEscherichia coli70S ribosome with a full set of recombinant ribosomal proteins
Author(s) -
Ryo Aoyama,
Keiko Masuda,
Masaru Shimojo,
Takashi Kanamori,
Takuya Ueda,
Yoshihiro Shimizu
Publication year - 2021
Publication title -
journal of biochemistry/the journal of biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.28
H-Index - 115
eISSN - 1756-2651
pISSN - 0021-924X
DOI - 10.1093/jb/mvab121
Subject(s) - ribosome , 50s , ribosomal protein , ribosomal rna , 30s , eukaryotic ribosome , escherichia coli , recombinant dna , eukaryotic large ribosomal subunit , protein subunit , biology , computational biology , chemistry , 18s ribosomal rna , biochemistry , rna , gene
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.