PROJECT SUMMARY Many biomedically significant proteins, including antibodies, cytokines, anticoagulants, blood clotting factors, and others are glycoproteins. Thus, there is a high demand for systems that can be used to produce recombinant glycoproteins for basic biomedical research and direct clinical applications. Unfortunately, few currently available recombinant protein production systems can produce higher eukaryotic glycoproteins with authentic, relatively homogeneous carbohydrate side chains at relatively low cost. The long-term objective of this proposal is to genetically engineer the silkworm, Bombyx mori, as a system that can fulfill these requirements for recombinant glycoprotein production. Numerous studies have shown that the silkworm silk gland, which has evolved for millions of years as a highly efficient silk protein production and secretion organ, can be engineered to efficiently produce and secrete recombinant proteins. However, transgenic silkworms have not yet been effectively used for recombinant glycoprotein production because the endogenous protein glycosylation pathways of the silk gland cannot properly glycosylate foreign, higher eukaryotic glycoproteins. The proposed research seeks to develop the silkworm as a novel system for recombinant human glycoprotein production by creating transgenic silkworms encoding a set of higher eukaryotic enzymes needed to “humanize” the native silk gland protein N-glycosylation pathway and recombinant human N-glycoproteins of interest. Each transgene will be placed under the control of the tissue-specific Ser1 promoter to target its expression to the middle silk gland. To our knowledge, there is only one prior report of the effective genetic engineering of a protein glycosylation pathway in any multicellular animal, including B. mori. We will build upon our initial success of glycoengineering the protein N-glycosylation pathway of B. mori to significantly advance the use of the silk gland as a bioreactor for recombinant glycoprotein production and secretion. This will have a net positive effect on silk industries in both developed and underdeveloped countries worldwide, allowing value added products important for human health to be produced in an economically feasible manner in addition to the basic silk fiber, thereby significantly increasing the value of this important biomanufacturing platform. The Jarvis and Fraser laboratories have a demonstrated ability to perform this research as shown by their publication records. In addition, they have been productively collaborating on related projects for the past 15 years, generating a significant amount of relevant preliminary data. The complementary skills available in these two labs, their established working relationship, and the preliminary data obtained to date strongly suggest the proposed research can be successfully completed.