Baculovirus Expression Vectors are widely used for high-level protein production with applications in medical therapeutics, diagnostics, vaccines, and drug discovery. The power of the baculovirns system is due to the viral RNA polymerase. This is a simple four-subunit complex that has, in addition to its catalytic activity, the ability to specifically recognize viral promoters and to process RNAs at both the 5' and 3' ends. The goal of this proposal is to develop baculovirus RNA polymerase as a commercial tool for the production of capped and polyadenylated transcripts. There are no commercially available enzymes for simultaneous transcription and processing of RNAs in vitro. Several companies sell kits for the production of capped RNAs. but this requires the use of cap analog. Baculovirus RNA polymerase should produce capped RNAs more cheaply and efficiently, and would also polyadenylate the RNAs which should provide increased stability and efficiency of translation.
Our specific aims are to: 1) Develop a bacterial expression system for baculovirus RNA polymerase; 2) Design a purification scheme that yields enzyme that is 95% pure and 95% active; and 3) Analyze the promoter specificity of baculovirus RNA polymerase. In Phase II of the program, we will develop commercial products, including an improved coupled transcription-translation system and a diagnostic kit for protein truncation assays.
Baculovirus RNA polymerase will be developed as a commercial tool for the production of capped and polyadenylated transcripts. This will be useful for researchers needing RNAs with increased stability and improved translational efficiency in vivo and in vitro. In addition, baculovirns RNA polymerase should offer improved thoughput for medical diagnostic assays based on coupled transcription/translation systems like the protein truncation test.
