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영어

Affinity tags have become indispensable tools for protein expression and purification, and tobacco etch virus (TEV) protease is widely used as a cleavage enzyme to produce recombinant proteins which are expressed as a fusion protein. 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, we designed a mutant TEV protease(T17S,N68D, I77V, S219V) was fully codon-optimized based on Oryza sativa codon usage with mutations that resist autoproteolytic inactivation and improve the solubility. The resulting sTEV 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. sTEV protease mRNA and protein expression in transgenic rice suspension cells during sugar starvation under the control of RAmy3D promoter were confirmed by Northern and Western blot analyses (This study was supported by Technology Development Program (108166-03-1-HD110) of Ministry for Food, Agriculture, Forestry and Fisheries of the Republic of Korea).