Molecular cloning and characterization of the human RNase kappa, an ortholog of Cc RNase.
A novel protein family, designated hereafter as RNase kappa (kappa) family, has been recently introduced with the characterization of the specific Cc RNase, isolated from the insect Ceratitis capitata. The human ortholog of this family consists of 98 amino acids and shares > 98% identity with its mammalian counterparts. This RNase is encoded by a single-copy gene found to be expressed in a wide spectrum of normal and cancer tissues. The cDNA of the human ribonuclease has been isolated and subcloned into a variety of prokaryotic expression vectors, but most efforts to express it caused a severe toxic effect. On the other hand, the expression of the human RNase by the use of the methylotrophic yeast Pichia pastoris system resulted in the production of a highly active recombinant enzyme. Using a 30-mer 5'-end-labeled RNA probe as substrate, the purified enzyme seems to preferentially cleave ApU and ApG phosphodiester bonds, while it hydrolyzes UpU bonds at a lower rate. Based on amino acid sequence alignment and substrate specificity data, as well as the complete resistance of the recombinant protein to the placental ribonuclease inhibitor, we concluded that the human RNase kappa is a novel endoribonuclease distinct from other known ribonucleases.
Economopoulou
Marie-Angela I
MA
University of Athens, Faculty of Biology, Department of Biochemistry and Molecular Biology, Panepistimioupolis, 15701, Athens, Greece.
Fragoulis
Emmanouel G
EG
Sideris
Diamantis C
DC
D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D017124:Conserved Sequence; D018076:DNA, Complementary / Q000302:isolation & purification; D004722:Endoribonucleases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D012260:Ribonucleases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D016415:Sequence Alignment; D013379:Substrate Specificity
D018076:DNA, Complementary; D012333:RNA, Messenger; D004722:Endoribonucleases; C525216:RNase kappa, human; D012260:Ribonucleases