Animal 2-5A-dependent RNases and encoding sequences therefor

US5877019A · kind A · utility

Assignee

• The Cleveland Clinic Foundation · US

Inventors

• Robert H. Silverman · Shaker Heights, US
• Bret A. Hassel · Woodbine, US
• Aimin Zhou · Solon, US

Key dates

• Filing date: Aug 21, 1996
• Grant date: Mar 2, 1999
• Expiry date: Aug 21, 2016

Classification

• Technology area (CPC C): Chemistry; Metallurgy
• CPC primary: C12N9/22
• WIPO field: Biotechnology
• WIPO sector: Chemistry

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.