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Affinity tags have become indispensable tools for protein expression and purification, and tobacco etch virus (TEV) protease emerge as a useful reagent with wide application in the cleavage of recombinant fusion proteins. Upon expression of TEV protease in Escherichia coli expression system, a recombinant protein was produced that exhibited proteolytic activity toward EGFP-hTNF-a (fusion protein expressed in E. coli) in vitro. In order to high-level expression in plant, TEV protease was fully codon-optimized based on Oryza sativa codon usage with mutations that resist autoproteolytic inactivation and improve the solubility. The resulting
shTEV was subcloned into the plant expression vector and then introduced into rice callus via particle bombardment-mediated transformation. Fourteen transgenic lines were obtained and the transgene insertion was confirmed by genomic DNA PCR. shTEV protease mRNA and protein
expression in transgenic rice suspension cells during sugar starvation under the control of RAmy3D promoter were confirmed by Northern and SDS-PAGE analyses (This Study was supported by Technology Development Program for Agriculture and Forestry of MIFAFF).