Expression of Human Protein C in Transgenic Tobacco
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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.
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