CLONING, OVEREXPRESSION AND PURIFICATION OF RECOMBINANT TOBACCO ECTH VIRUS PROTEASE (TEVp) IN Echerichia coli

Abstract

One of the challenges for recombinant protein technology is the expression and purification of recombinant proteins in host cells; certain proteins are not expressed or expressed in the inactive forms which do not have their desired biological functions, even toxic to their hosts. To overcome this drawback, the target protein is usually expressed as a fusion protein in which target protein was fused to other protein such as thioredoxin (TRX), Glutathione G-Trans (GST), NusA, Maltose

binding protein (MBP), histidine (His), etc. However, the fusion protein would cause a limited application of recombinant protein as it may cause false negative/positve results of diagnostic kits, or allergy when they are used as pharmaceutical or vaccine. Therefore, the fusion protein should be removed from the target protein by treatment with specific protease. Due to the high specificity, TEV protease (TEVp), a protease from Tobacco Etch Virus (TEV), was selected for this purposes. Gene encoding for TEVp was amplified by RT-PCR, cloned into the expression vector pET21-a(+) to yield recombinant plasmid pETEVp and then transformed into E. coli strain BL21 (DE3) to overexpress the target protein. The results indicate that, recombinant TEVp expressed as insoluble form at temperatures of 37, 30 and 25 °C, but at 18 °C it exists mainly in soluble form. Optimal conditions for the expression of recombinant proteins have also been determined. Recombinant TEVp was successfully purified by Ni-NTA affinity chromatography column under native condition with high purity and showed specific activity to its substrate.