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

Backgrounds: Peptide tags are protein sequences that are used in recombinant proteins mainlyin order to increase the solubility or facilitate the purification of the proteins. Generally, the usedtags need to be removed accurately and appropriately after the production and purification of therecombinant proteins. Accordingly, in the present study, a plasmid was constructed thatexclusively encoded TEV protease using arabinose as the inducer.Materials and Methods: The constructed final vector was pBAD/GST.TEV/TT/Neo-Kan/p۱۵AOri, which was briefly called pBAD/GST. Also, the accuracy of all cloned sequenceswas confirmed by DNA sequencing. Finally, pBAD/GST plasmid was used to transform ShuffleT۷ Express E. coli cells and the expression of soluble TEV protease was confirmed using SDSPAGE.Results: After induction of the recombinant bacterial cells by defined concentrations ofarabinose at ۳۰ °C for ۴ h, an expected TEV protein with size of ۵۵ KDa was successfullyproduced. The production of the protein was confirmed by observing the protein band using SDSPAGE. Protease function of TEV inside the bacterial cells was verified by the cleavage of fusionprotein TRX IGF۱ produced in shuffle E. coli cells. For this, at first bacterial cells are induced byappropriate concentration of IPTG in order to express TRX IGF۱. After ۱ h, arabinose was addedto the media and the culture of the induced cells was continued for ۶ h. Total soluble fraction aswell as inclusion bodies were isolated and analyzed using SDS PAGE. The results demonstratedthat TEV protease cleaved the fusion protein in soluble fraction and inclusion bodies. Finally,purification was performed with Nika resin, which showed an IGF ۱ band.Conclusion: In this study, a new subspecies of Shuffle T۷ Express E. coli was obtained bytransformation of an expression plasmid, which could produce the soluble and active TEVprotease. This protease could cleave its specific site between TRX and IGF-۱ in the host bacterialcells and separate TRX tag from the recombinant IGF-۱, which was expressed by an independentpET vector. Therefore, by this approach there is no need to digest the recombinant fusion proteinafter extraction from the bacteria. Indeed, in vivo digestion of recombinant fusion protein canhave a significant effect on facilitating and reducing the costs of downstream purificationprocess.

کلیدواژه ها:

TEV protease ، Protein tag ، Recombinant protein ، Fusion protein