The purpose of this initiative is to develop a novel technology for the expression of authentic glycoproteins. Many proteins become post-translationally modified during the secretory process. These modifications are extremely important because they may alter the proteins'physical and chemical properties, folding, stability, and activity. These glycosylated proteins are often the targets of drug discovery. Therefore, it is highly desirable to develop an expression system that is fast and highly productive for authentic glycosylated proteins. We propose here to develop a unique mammalian expression system by introducing an RNA dependent RNA polymerase derived from Semliki Forest Virus to amplify the transgene RNA and provide an extra step in the natural transcription- translation process of protein production. The massive amplification of the target gene RNA makes the expression system highly efficient and reliable. Our expression system will inherit the intrinsic replication function of viral RNA dependent RNA polymerase and meanwhile, no viral structure protein or its RNA is involved in our system. Therefore, it is ultimately a safe system. With this system we can produce authentic glycosylated proteins for research, drug discovery and preclinical, and eventually, therapeutic glycosylated proteins and vaccine as well. PUBLIC HEALTH RELEVANCE: Most of the recombinant protein drugs are modified in the cell after being synthesized. It is necessary to make authentically modified proteins for drug development, drug production and disease related research. However, production of the authentically modified protein is a bottleneck for the drug development and production. In this proposal, we presented a novel and unique approach to design a mammalian expression system that is capable to produce milligram quantity of authentically modified proteins. Taken advantage of the RNA dependent RNA polymerase (RdRP) from Semliki Forest virus, the recombinant protein gene will be amplified at RNA stage. The massive amplified RNA will take over the protein synthesis machinery and effectively produce large amount of recombinant protein. The novel system will meet the demand of drug development and preclinical studies.