Sequence, overproduction and purification of Vibrio proteolyticus ribosomal protein L18 for in vitro and in vivo studies.
A strategy suggested by comparative genomic studies was used to amplify the entire Vibrio proteolyticus (Vp) gene for ribosomal protein L18. Vp L18 and its flanking regions were sequenced and compared with the deduced amino acid (aa) sequences of other known L18 proteins. A 26-aa residue segment at the carboxy terminus contains many strongly conserved residues and may be critical for the L18 interaction with 5S rRNA. This approach should allow rapid characterization of L18 from large numbers of bacteria. Both Vp L18 and Escherichia coli (Ec) L18 were overproduced and purified using a T7 expression vector which fuses an N-terminal peptide segment (His-tag) containing 6 histidine residues to the recombinant protein. The purified fusion proteins, Vp His::L18 and Ec His::L18, were both found to bind to either the Vp 5S or Ec 5S rRNAs in vitro. Vp His::L18 protein was also shown to incorporate into Ec ribosomes in vivo. This His-tag strategy likely will have general applicability for the study of ribosomal proteins in vitro and in vivo.
Setterquist
R A
RA
Department of Biochemical and Biophysical Sciences, University of Houston, TX 77204-5934, USA.
Smith
G K
GK
Oakley
T H
TH
Lee
Y H
YH
Fox
G E
GE
D000595:Amino Acid Sequence; D017123:Bacteriophage T7 / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics; D006639:Histidine; D008969:Molecular Sequence Data; D010455:Peptides; D011485:Protein Binding; D012341:RNA, Ribosomal, 5S / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012269:Ribosomal Proteins / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014733:Vibrio / Q000235:genetics*
D010455:Peptides; D012341:RNA, Ribosomal, 5S; D011993:Recombinant Fusion Proteins; D012269:Ribosomal Proteins; C029609:ribosomal protein L18; C033223:polyhistidine; D006639:Histidine