Purification and characterization of potato ribonuclease P
Tóm tắt
Ribonuclease P (RNase P), a ribonucleo-protein endoribonuclease, responsible for 5′ maturation of precursor tDNA, is well characterized in bacteria, yeast and human, but not in plant. Attempt has been made to partially purify and characterize nuclear RNase P from potato. cDNAs encoding two putative protein subunits of potato ribonuclease P (RNase P), StPop5 and StRpp25, were picked from potato EST library based on homology with respective human RNase P protein subunits. Both the cDNAs, 435 bp long StPop5 and 765 bp long StRpp25, were RT-PCR amplified, cloned and sequenced. StPop5 exhibited 46 % nucleotide sequence similarity to the hPop5 sequence. The deduced amino acid sequence of StPop5 had 23 % identity and 35 % similarity to hPop5. Both hRpp25 and StRpp25 had 46 % nucleotide sequence homology, and 17 % identity and 27 % similarity at a length of 271 amino acids. The molecular masses of purified 6× His-tagged recombinant StPop5 and StRpp25 proteins were 18 kDa and 33 kDa respectively. Potato nuclear RNase P was partially purified from leaves employing DEAE-Sephacel anion-exchange chromatography, and from floral buds employing DEAE-Sephacel and HiTrap Q anion-exchange chromatography, ammonium sulfate precipitation and gel filtration chromatography. Immunoprecipitation with polyclonal antisera, raised against recombinant StPop5 and StRpp25, demonstrated association of these two proteins with floral bud RNase P activity but not with leaf RNase P activity.
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