Research Project
3507980
DESCRIPTION (Adapted from applicant's abstract): Numerous expression systems exist for the bioproduction of proteins in both prokaryotic and eukaryotic cells. Each system has its own set of advantages as well as shortcomings. The need remains for a mammalian cell expression system that can achieve high-level gene expression, correctly process proteins of mammalian origin, and is economical and easy to use. The aim is of this proposal is to establish a mammalian cell expression system capable of rapid and high level production of specified proteins. To this end, the use of regulatory components from the E. coli lactose operon to regulate the expression of bacteriophage T7 RNA polymerase in a single vaccinia virus-based vector system was investigated. The requirement to control T7 RNA polymerase expression was necessary since previous attempts to develop such a single virus system without regulation were unsuccessful, presumably due to the high transcriptase activity and processibility of the phage RNA polymerase causing interference with endogenous viral transcription or replication. In Phase I, a single recombinant vaccinia virus harboring both T7 RNA polymerase and T7 promoter-controlled target gene sequences under regulatory control of genetic elements from the lactose operon was successfully constructed. Induction of this system led to a higher level of target gene expression than that obtained previously using a two virus system. In Phase II, the expression parameters for this system will be further optimized. This system will be adapted to pilot-level bioreactor instrumentation leading to the bioproduction of several proteins of interest. New strategies to further refine and expand the utility of this system will be evaluated for the commercial production of proteins.
