Expression of Human Protein C in Transgenic Tobacco

Abstract

Human Protein C (hPC) is a vitamin K-dependent serine protease that has a critical role in the naturally-occurring anticoagulant pathway. Upon activation of the zymogen by thrombin at the endothelial cell surface, the active form of hPC has anticoagulant activity in hemostasis due to its ability to inactivate factors Va and VIIIa. For biological activity, hPC requires several post-translational modifications including proteolytic cleavage, disulfide bond formation, b-hydroxylation, g-carboxylation, and N-linked glycosylation. Plants have the eukaryotic protein modifying mechanisms required for many human proteins and may provide a safe, cost-effective system for producing hPC on a large-scale basis. Tobacco (Nicotiana tabacum L.) is particularly well suited for use as a bioreactor for high-value recombinant proteins. Tobacco is one of the easiest plants to transform, it is an excellent biomass producer and can produce up to a million seeds from a single genetically engineered plant. Previous attempts to produce hPC in tobacco were limited by expression levels. The overall goal of the research was to develop transgenic plants that express hPC at higher levels. A cDNA encoding hPC was fused to an enhanced constitutive 35S promoter (35SDE ) and introduced into a plant transformation vector. The hPC construct was introduced into tobacco leaf disks using Agrobacterium tumefaciens-mediated transformation, and 30 transgenic plants were generated. Stable integration of the hPC gene construct into the tobacco genome and transgene copy number were determined by genomic Southern hybridization and segregation analyses. The majority of transgenic plants expressed the hPC transgene based on RNA analyses by northern hybridization. Plants utilizing the enhanced 35S promoter had equivalent levels of expression to previously generated hPC-containing plants. A variety of polyclonal and monoclonal antibodies raised against hPC were tested for detection of hPC standards and tobacco-synthesized hPC by western immunoblotting. Novel proteins in the size range of hPC heavy chain cross-reacted with anti-heavy chain hPC antibodies in 35SDE:hPC plants. Thus, plants may be capable of synthesizing hPC and proteolytically processing it to light and heavy chains. Although further experiments will be required to confirm the identity of these putative hPC proteins in tobacco, these result suggest that analyses of hPC expressed in plants have been limited by effective tools for detecting the hPC gene product rather than expression levels determined by the transgene promoter.