Introduction: Peptide tags are protein sequences that are used in recombinant proteins mainly in order to increase the solubility or facilitate the purification of recombinant proteins. Generally, the used tags need to be removed accurately and appropriately after the production and purification of recombinant proteins. Using the proteases such as TEV proteases is one way to remove the tags. This protease is produced by Tobacco etch virus (TEV). Simultaneous production of this protease in the target recombinant protein-producing bacterium causes the isolation of solubilizing tags from target proteins. The aim of this study is the construction a TEV protease-producing expression vector and its transform to an appropriate species of E. coli, which is used to produce recombinant protein. In this way, a new subspecies is constructed in which the structure producing the TEV protease in a controlled manner produces appropriate amounts of TEV protease.
Methods: First, the specific primers that contained suitable restriction enzymes at 5'ends were designed for amplification of GST.TEV fragment form pGEX-4T1 vector.Then it was cloned into the T vector as cloning vector and subsequently cloned into the expression vector pBAD-GIIIA. After that, the terminator fragment was amplified from pBADgIIIA vector by proper primers and cloned downatream of GST.TEV fragment in the pBAD-GIIIA vector. The NEO.KNA fragment was amplified from EGFPC1 vector by proper primers and was cloned in pBAD-GIIIA downstream of terminator fragmentvector. At the end, the p15A Ori fragment was amplified from the pG.TF2 vector by appropraite primers and subcloned into the pBAD-GIIIA downstream of NEO.KAN fragment. All fragments was are verified by sequencing.
Results: In this study, a new subspecies of E. coli is obtained with construction of recombinant vector which could produces the soluble active TEV protease.
Conclusion: This protease could remove dissolution tags from recombinant proteins with suitable digestion sites. In this situation, the dissolution tag used upstream or downstream of the recombinant protein and removed in the host bacterial cells that can have a significant effect in facilitating and reducing the costs of downstream purification processes.
Keywords: TEV protease, Protein tag, Recombinant protein, Fusion protein, Expression vector, E. coli