title: Soluble expression of aggregating proteins by covalent coupling to the ribosome.
abstract: Ribosomes are extremely soluble ribonucleoprotein complexes. Heterologous target proteins were fused to ribosomal protein L23 (rpL23) and expressed in an rpL23 deficient Escherichia coli strain. This enabled the isolation of 70S ribosomes with covalently bound target protein. Isolation of recombinant proteins from 70S ribosomes was achieved by specific proteolytic cleavage followed by efficient removal of ribosomes by centrifugation. By this procedure we isolated active green fluorescent protein, streptavidin (SA), and murine interleukin-6 (mIL-6). Approximately 500microg of each protein was isolated per gram cellular wet weight. By pull-down assays we demonstrate that SA covalently bound to the ribosome binds d-biotin. Ribosomal coupling is therefore suggested as a method for the investigation of protein interactions. The presented strategy is in particular efficient for the expression, purification, and investigation of proteins forming inclusion bodies in the E. coli cytoplasm.
authors.affiliation: Laboratory of BioDesign, Department of Molecular Biology, Aarhus University, Gustav Wieds Vej 10 C, DK-8000 Aarhus C, Denmark.
authors.forename: Hans Peter
authors.initials: HP
authors.lastname: Sørensen
authors.forename: Janni Egebjerg
authors.initials: JE
authors.lastname: Kristensen
authors.forename: Hans Uffe
authors.initials: HU
authors.lastname: Sperling-Petersen
authors.forename: Kim Kusk
authors.initials: KK
authors.lastname: Mortensen
mesh_terms: D000818:Animals; D001710:Biotin / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D015850:Interleukin-6 / Q000235:genetics / Q000378:metabolism; D051379:Mice; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000235:genetics / Q000378:metabolism*; D012270:Ribosomes / Q000235:genetics / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; D015850:Interleukin-6; D011993:Recombinant Fusion Proteins; D012269:Ribosomal Proteins; D001710:Biotin