Abstract
Because of its stringent sequence specificity, tobacco etch virus (TEV) protease is widely used to remove fusion tags from recombinant proteins. Due to the poor solubility of TEV protease, many strategies have been employed to increase the expression level of this enzyme. In our work, we introduced a novel method to produce TEV protease by using visible superfolder green fluorescent protein (sfGFP) as the fusion tag. The soluble production and catalytic activity of six variants of sfGFP-TEV was examined, and then the best variant was selected for large-scale production. After purified by Ni-NTA affinity chromatography and Q anion exchange chromatography, the best variant of sfGFP-TEV fusion protease was obtained with purity of over 98% and yield of over 320 mg per liter culture. The sfGFP-TEV had a similar catalytic activity to that of the original TEV protease. Our research showed a novel method of large-scale production of visible and functional TEV protease for structural genomics research and other applications.
Highlights
Nowadays, it has been a popular way to fuse target proteins with various tags to facilitate expression and purification
In order to maximize the expression level of the recombinant sf green fluorescent protein (GFP)-tobacco etch virus (TEV) proteases, we first synthesized the superfolder green fluorescent protein (sf GFP) gene according to the synonymous codon choice which is optimal for the Escherichia coli translational system
The sf GFP-TEV coding sequence was cloned to the pET derived vector pT7His which possesses the strong bacteriophage T7 promoter, ensuring the high level expression of target protein
Summary
Because of its stringent sequence specificity, tobacco etch virus (TEV) protease is widely used to remove fusion tags from recombinant proteins. We introduced a novel method to produce TEV protease by using visible superfolder green fluorescent protein (sf GFP) as the fusion tag. The soluble production and catalytic activity of six variants of sf GFP-TEV was examined, and the best variant was selected for large-scale production. After purified by Ni-NTA affinity chromatography and Q anion exchange chromatography, the best variant of sf GFP-TEV fusion protease was obtained with purity of over 98% and yield of over 320 mg per liter culture. Our research showed a novel method of large-scale production of visible and functional TEV protease for structural genomics research and other applications
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