tRNA-assisted overproduction of eukaryotic ribosomal proteins.
Structural studies of eukaryotic ribosomes are complicated by the tendency of their constituent proteins to be expressed at very low levels in Escherichia coli. We find that this is mainly due to their exceptionally high content of AGA/AGG arginine codons, which are poorly utilized by the bacterial translational machinery. In fact, we could overcome this limitation by the combined use of a T7 RNA polymerase expression vector and a plasmid carrying the E. coli gene argU, which encodes the minor tRNA(Arg) species that reads AGA/AGG codons. In this system, five cytoplasmic ribosomal proteins from three different eukaryotic lineages (Saccharomyces cerevisiae S8, L13, and L14; Arabidopsis thaliana L13; and Homo sapiens L7) could be overexpressed to up to 50% of total bacterial protein and were purified to homogeneity in tens of milligrams amounts. The purification procedure simply involved metal affinity chromatography followed, in some cases, by an additional heparin chromatography step. Recombinant polypeptides bound RNA with high affinity (K(d) between 50 and 300 nM). This novel overexpression/purification strategy will allow the production of high amounts of most eukaryotic ribosomal proteins in a form suitable for structural and functional studies. Coupled with recently completed and ongoing whole-genome sequencing projects, it will facilitate the molecular characterization of the eukaryotic ribosome.
Dieci
G
G
Institute of Biochemical Sciences, University of Parma, Parma, I-43100, Italy.
Bottarelli
L
L
Ballabeni
A
A
Ottonello
S
S
D017360:Arabidopsis / Q000737:chemistry; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins; D006801:Humans; D024101:Mitochondrial Proteins; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D012313:RNA / Q000737:chemistry; D012347:RNA, Transfer, Arg / Q000235:genetics / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000737:chemistry; D029701:Saccharomyces cerevisiae Proteins; D014764:Viral Proteins
D029968:Escherichia coli Proteins; C489211:MRPL13 protein, S cerevisiae; D024101:Mitochondrial Proteins; D012347:RNA, Transfer, Arg; C053634:RPL7 protein, human; D012269:Ribosomal Proteins; D029701:Saccharomyces cerevisiae Proteins; D014764:Viral Proteins; C041174:ribosomal protein L14; C030254:ribosomal protein S8; C512844:rplL protein, E coli; D012313:RNA; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases