KSEF R&D Excellence: Development of a System for Producing Highly Purified Proteins on the Plant Surface

Development of a System for Producing Highly Purified Proteins on the Plant Surface. PI - George J. Wagner, Agronomy Department, University of Kentucky, Lexington KY 40546-0236 We have discovered a system which produces and accumulates proteins on tobacco and other plant leaf surfaces (SPPS, surface protein production system). We observe that SPPS causes accumulation of substantial amounts of previously uncharacterized proteins (phylloplanins) which we demonstrate inhibit disease caused by the fungus-like pathogen responsible for blue mold disease. We isolated the phylloplanin gene and its promoter (patents pending, R. Shepherd and G. Wagner) and used the promoter to drive the expression of animal and bacterial genes encoding reporter proteins. Practical use of SPPS would involve fusing the phylloplanin gene promoter to various target genes to create transgenic plants that secrete target proteins to the plant surface. Relatively pure proteins would be easily and very economically recovered from the surface without the need to macerate tissue and contaminate the product. The rationale is to determine if SPPS can provide an economically superior system to conventional systems for producing commercially used proteins (e.g., pharmaceutical cytokines, restriction enzymes, etc.). Since 85% of costs in producing recombinant proteins in plants are estimated to accrue during purification, SPPS has potential to drastically reduce production costs below any system now available. Our vision is that SPPS will extend our current exploitation of plant surface secretion of anti-fungal tobacco phylloplanins, which is the focus of a start-up company, PhylloTech, LLC. The work described here will not focus on enhancement of disease resistance, but rather on use of SPPS to produce recombinant proteins. Distinguishing features of SPPS are its potential for high yield, lowest purification costs, and its amenability to production of potentially toxic proteins. The specific objectives of this project are: 1) to determine if commercially important pharmaceutical proteins can be produced by SPPS, 2) to determine the production capacity of SPPS for various proteins (yield), 3) to determine the stability of accumulating protein with time, and 4) determine if a protein that cannot be prepared by overexpression in E. coli due its toxicity can be produced by SPPS. Actions to enable include the assessment of the scope of usefulness of SPPS by testing genes encoding various proteins from different organisms, quantification of protein production capacity, and assessment of protein stability during accumulation. Key Words: plant-protein-production-system, protein-pharmaceuticals, heterologousprotein-expression, tobacco, surface-secretion