Introduction: 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 p15A Ori, which is compatible with pBR322 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.
Methods: First, the specific primers that contained suitable restriction enzymes at the 5' ends were designed for amplification of GST tagged TEV coding fragment (GST.TEV) form pGEX-4T1 vector. Amplified GST.TEV was cloned into a T vector and subsequently sub-cloned into the expression vector pBAD-GIIIA, called pBAD/GST.TEV. The transcription terminator (TT) sequence was then amplified from pBAD-GIIIA vector by proper primers and sub-cloned downstream of GST.TEV fragment in the pBAD/GST.TEV. The resulted vector was named pBAD/GST.TEV/TT. Moreover, the coding sequence of neomycin-kanamycin phosphotransferase (Neo-Kan) was amplified from pEGFPC1 vector by the suitable primers and was sub-cloned in pBAD/GST.TEV/TT, downstream of transcription terminator that resulted pBAD/GST.TEV/TT/Neo-Kan. At the end, the p15A Ori segment was amplified from the pG.TF2 vector by designed primers and sub-cloned into pBAD/GST.TEV/TT/Neo-Kan downstream of Neo-Kan fragment. The constructed final vector was pBAD/GST.TEV/TT/Neo-Kan/p15AOri, which was briefly called pBAD/GST. Also, the accuracy of all cloned sequences was confirmed by DNA sequencing. Finally pBAD/GST plasmid was used to transform Shuffle T7 Express E. coli cells and the expression of soluble TEV protease was confirmed using SDS-PAGE. The activity of the recombinant TEV protease was evaluated in the Shuffle cells that were previously transformed by a pET32 vector expressing IGF-1 tagged with Thioredoxin (TRX).
Results: In this study, a new subspecies of Shuffle T7 Express E. coli was obtained by transformation of an expression plasmid, which could produce the soluble and active TEV protease
Conclusion: This protease could cleave its specific site between TRX and IGF-1 in the host bacterial cells and separate TRX tag from the recombinant IGF-1, which was expressed by an independent pET vector. Therefore, by this approach there is no need to digest the recombinant fusion protein after extraction from the bacteria. Indeed, in vivo digestion of recombinant fusion protein can have a significant effect on facilitating and reducing the costs of downstream purification process.
Keywords: TEV protease, Protein tag, Recombinant protein, Fusion protein, Shuffle T7 Express E. coli