An improved procedure for expression and purification of ribosomal protection protein Tet(O) for high-resolution structural studies.
Tetracycline (Tc) is a broad spectrum antibiotic that binds to the A site of the bacterial ribosome inhibiting delivery of aminoacyl-tRNA to the A site for productive protein biosynthesis. Tet(O) is in a class of the ribosomal protection proteins (RPPs) found in many pathogenic bacteria, that dislodges Tc from the A site of 70S ribosome to restore polypeptide elongation and confer Tc resistance to the bacteria. Considerable difficulty has been encountered in overexpressing and purifying Tet(O) from various Escherichia coli strains using lambdaPI, tac or T7 promoters. Here we report molecular cloning, overexpression of His-tagged Tet(O) in E. coli, an improved purification procedure and initial biochemical and biophysical characterization of His-tagged Tet(O).
Thakor
Nehal S
NS
Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alta., Canada T6G 2H7.
Wilson
Kevin S
KS
Scott
Paul G
PG
Taylor
Diane E
DE
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D002853:Chromatography, Liquid; D002942:Circular Dichroism; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D006160:Guanosine Triphosphate / Q000378:metabolism; D006868:Hydrolysis; D008027:Light; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012542:Scattering, Radiation; D012995:Solubility
D001426:Bacterial Proteins; D002352:Carrier Proteins; D017931:DNA Primers; D011994:Recombinant Proteins; C083139:Tet O resistance protein, Bacteria; D006160:Guanosine Triphosphate