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

Background and Aim : 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. 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-Kna resistance gene in the plasmid that allows the selection of transformed bacteria that already had pET vector with Amp resistance gene. Methods : First, the specific primers that contained suitable restriction enzymes at the ۵' ends were designed for amplification of GST tagged TEV coding fragment (GST.TEV) form pGEX-۴T۱ 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 pEGFPC۱ 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 p۱۵A Ori segment was amplified from the pG.TF۲ 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/p۱۵AOri, which was briefly called pBAD/GSTResults : In this study, a new subspecies of Shuffle T۷ 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-۱ in the host bacterial cells and separate TRX tag from the recombinant IGF-۱, which was expressed by an independent pET vector

کلیدواژه ها:

E. coli ، Expression vector ، Fusion protein ، Protein tag Recombinant protein ، TEV protease.