Recombinant method for making RSV reverse transcriptases and mutants thereof
AssigneeInvitrogen Corporation
InventorsGary F Gerard, Michael D Smith, Deb K Chatterjee
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Abstract
A method of producing a Rous Sarcoma Virus reverse transcriptase (RSV RT) by expressing one orniore nucleic acid sequences encoding one or more subunits of RSV RT in a eukaryotic host cell and culturing the host cell under conditions sufficient to produce the recombinant RSV RT. The resulting RSV RT has a specific activity of between about 30,000 and 150,000 units per milligram and is suitable for methods including RT-polyinerase chain reaction (RT-PCR).
Claims62
A method of producing a Rous Sarcoma Virus (RSV) reverse transcriptase having a RNA-dependent DNA polymerase specific activity of 30,000 units per milligram to 150,000 units per milligram, said method comprising
- (a) obtaining a eukaryotic host cell comprising one or more nucleic acid sequences encoding one or more subunits of RSV reverse transcriptase;
- (b) culturing said host cell under conditions sufficient to produce said RSV reverse transcriptase;
- and
- (c) isolating or purifying said reverse trascriptase thereby obtaining an RSV reverse transcriptase having a RNA-dependent DNA polymeraso specific activity of 30,000 units per milligram to 150,000 units per milligram.
A method of producing a Rous Sarcoma Virus (RSV) reverse transcriptase having a RNA-dependent DNA polymerase specific activity of 30,000 units per milligram to 150,000 units per milligram or a mutant RSV reverse transcriptase having reduced RNase H activity, said method comprising
- (a) obtaining a eukaryotic host cell comprising one or more nucleic acid sequences encoding one or more mutant subunits of RSV reverse transcriptase having reduced RNase H activity relative to the wild-type reverse transcriptase;
- (b) culturing said host cell under conditions sufficient to produce said mutant RSV reverse transcriptase;
- and
- (c) isolating or purifying said reverse trascriptase thereby obtaining a mutant RSV reverse transcriptase having a RNA-dependent DNA polymerase specific activity of 30,000 units per milligram to 150,000 units per milligram.
Dependencies are inferred from the claim text.