A single-step method for purification of active His-tagged ribosomes from a genetically engineered Escherichia coli

Abstract

With the rapid development of the ribosome field in recent years a quick, simple and high-throughput method for purification of the bacterial ribosome is in demand. We have designed a new strain of Escherichia coli (JE28) by an in-frame fusion of a nucleotide sequence encoding a hexa-histidine affinity tag at the 3′-end of the single copy rplL gene (encoding the ribosomal protein L12) at the chromosomal site of the wild-type strain MG1655. As a result, JE28 produces a homogeneous population of ribosomes (His)6-tagged at the C-termini of all four L12 proteins. Furthermore, we have developed a single-step, high-throughput method for purification of tetra-(His)6-tagged 70S ribosomes from this strain using affinity chromatography. These ribosomes, when compared with the conventionally purified ones in sucrose gradient centrifugation, 2D-gel, dipeptide formation and a full-length protein synthesis assay showed higher yield and activity. We further describe how this method can be adapted for purification of ribosomal subunits and mutant ribosomes. These methodologies could, in principle, also be used to purify any functional multimeric complex from the bacterial cell.

Organization of Ribosomes and Nucleoids in Escherichia coli Cells during Growth and in Quiescence

“…1A shows the scheme for fusing mCherry tag to the ribosomal protein (r-protein) L9. The termination codon of the gene rplI (encoding L9) on the chromosome of E. coli MG1655 (WT) was replaced by a linear DNA containing the DNA sequence coding for the red fluorescent protein mCherry (32) and kanamycin resistance cassette (Kan R ) using -Red recombineering (33,34). The recombinants with L9-mCherry fusion were selected against kanamycin and verified by PCR and sequencing.…”

Investigation of protein–RNA interactions by mass spectrometry—Techniques and applications

“…His-tag, GSTtag etc.-can be fused to a protein within the protein-RNA complex of interest (for an example see Ref. [27]). …”

Omics in Aid of Host-pathogen Interaction Studies: A Bacterial Perspective

“…In principle, similar strategies making use of ribosome tagged bacterial strains [107] would permit selective isolation of bacterial ribosomes when studying the bacterial translatome in a host cell specific context. Moreover, as in the case of the multi RNA-seq strategies described above, it is still highly preferable to enrich for the desired subset of bacterial transcripts, especially in the context of newly translated proteins.…”