Nucleic Acids Research, Vol 26, Issue 13 3090-3096, Copyright © 1998 by Oxford University Press
S Niranjanakumari, JC Kurz and CA Fierke
Ribonuclease P is a ribonucleoprotein complex that catalyzes the essential
5' maturation of all precursor tRNA molecules. The protein component both
alters the conformation of the RNA component and enhances the substrate
affinity and specificity. To facilitate biochemical and biophysical
studies, the protein component of Bacillus subtilis ribonuclease P (RNase
P) was overproduced in Escherichia coli using the native amino acid
sequence with the initial 20 codons optimized for expression in E.coli . A
simple purification procedure using consecutive cation exchange
chromatography steps in the presence and absence of urea was developed to
purify large quantities of P protein without contaminating nucleic acids.
The identity of the recombinant protein as a cofactor of RNase P was
established by its ability to stimulate the activity of the RNA component
in low ionic strength buffer in a 1:1 stoichiometry. Circular dichroism
studies indicate that P protein is a combination of alpha-helix and
beta-sheet secondary structures and is quite stable, with a T m of 67
degrees C. The described methods facilitated the large scale purification
of homogeneous, RNA-free P protein required for high resolution
crystallographic analyses and may be useful for the preparation of other
RNA binding proteins.
ARTICLES
Expression, purification and characterization of the recombinant ribonuclease P protein component from Bacillus subtilis
Department of Biochemistry, Box 3711, Duke University Medical Center, Durham, NC 27710, USA.
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