Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Insect cell – per se
Patent
1996-08-21
1999-03-02
Grimes, Eric
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Insect cell, per se
435199, 435325, C12N 516, C12N 922
Patent
active
058770194
ABSTRACT:
2-5A-dependent RNase, an endoribonuclease that requires 5'-phosphorylated 2',5'-linked oligoadenylates (2-5A), functions in the molecular mechanism of interferon action. Recombinant, 2-5A-dependent RNase was expressed to high levels (at least 10% of the soluble protein) in insect cells by infecting with baculovirus containing human cDNA to 2-5A-dependent RNase. In contrast, there was no 2-5A-dependent RNase present in control insect cells infected with nonrecombinant baculovirus. The purified, recombinant enzyme eluted from a gel-filtration column as a monomer that showed potent and highly specific, 2-5A-dependent RNase activity. Precise activitor requirements were determined using the purified enzyme of a variety of 2',5'-linked oligonucleotides. The activated enzyme was capable of cleaving both poly(rU) and, to a lesser extent, poly(rA) but not poly(rC), poly(rG), or poly(dT. Interestingly, poly(rU) was cleaved to a series of discrete products ranging between 5 and 22 nucleotides in length. Furthermore, whereas manganese and magnesium stimulated 2-5A-dependent RNase activity, the enzyme was capable of cleaving RNA in the absence of divalent cations.
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Hassel Bret A.
Silverman Robert H.
Zhou Aimin
Cleveland Clinic Foundation
Grimes Eric
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