Creating an Efficient Strain for Purity of TEV-Labeled Recombinant Proteins

Peptide tags are protein sequences that are used in recombinant proteins mainly in order to increase the solubility or facilitate the purification of the proteins. Generally, the used tags need to be removed accurately and appropriately after the production and purification of the recombinant proteins. Using the some proteases such as TEV protease is a common method to remove the fusion tags. This protease is a highly sequence-specific cysteine protease, produced by Tobacco etch virus (TEV) and considered as one of the best endpeptidases for removing of the tags. We hypothesized that by induction of the bacterial cells to produce this protease at an appropriate time after production of the target recombinant protein, it is possible to digest and separate the tag from the target protein when the function of dissolution tag to improve recombinant protein solubility is completed. Accordingly, in the present study, a plasmid was constructed that exclusively encoded TEV protease using arabinose as the inducer. This vector was independent of the vector used to express the recombinant protein. In this plasmid, TEV encoding sequence was located under the BAD promoter, and p۱۵A Ori, which is compatible with pBR۳۲۲ Ori in pET expression vectors was used. We also used Neo-Kan resistance gene in the plasmid that allows the selection of transformed bacteria that already had pET vector with Amp resistance gene. This approach ensures that TEV encoding vector and pET vector expressing the recombinant protein to be compatible and can remain in the bacterial cells simultaneously. Following transformation of constructed TEV plasmid to E. coli, a new subspecies is acquired in which TEV protease is expressed by induction with arabinose that results in controlled production of TEV protease at an appropriate time to ensure that removal of the tag is taken place after completion of folding of recombinant protein.

Keywords:

TEV protease , Protein tag , Recombinant protein , Fusion protein , Shuffle T۷ Express E. Coli