Bacterial production and purification of human His-tagged IL-1RA and its digestion with TEV protease
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Sanaz Sedghi Esfahani,1 Pendar Shojaei Kojouri,2 Kianoush Dormiani,3,*
1. Department of Molecular Biotechnology, Cell Science Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran
2. Department of Molecular Biotechnology, Cell Science Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran
3. Department of Molecular Biotechnology, Cell Science Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran
- Introduction: Cytokines are small cell-signaling proteins which secreted by some cells like monocytes and macrophages. IL-1 family consists of 11 members including Interleukin-1 α (IL 1α), Interleukin-1 β (IL 1β) and IL 1 receptor antagonist (IL 1RA). IL-1 is a potent pro-inflammatory cytokine that activates the immune responses against infection and inflammation by stimulating lymphocytes B and T. IL 1RA is a natural inhibitor of IL 1α and IL 1β and blocks the IL-1 receptor type I, in competition with IL-1α or IL-1β. Considering, IL-1RA is unable to recruit the second receptor subunit IL-1 Receptor accessory protein (IL-1Racp), therefore the signaling pathways are not activated. The recombinant form of this protein is now available in the pharmaceutical market called Anakinra. This medicine can reduce the effects of the autoimmune diseases and also moderate inflammatory responses in patients with rheumatoid arthritis and infectious diseases. Based on the previous studies, this protein is produced mostly as insoluble form in E. coli. Accordingly, the present study was designed to construct an expression vector containing the coding sequence of human IL-1RA derived by T7 promoter into pET15b. Besides, an expression cassette of thioredoxin driven by T7 promoter was also inserted at downstream of IL-1RA cassette (TrxA). His-tag and TEV recognition site were also inserted upstream of the IL-1RA coding sequence. The recombinant IL-1RA protein in SHuffle T7 express E. coli was successfully produced into soluble form under an optimized induction condition 30°C, 4h and 0.5 mM IPTG. The biological function of this recombinant protein will evaluated in the future. The purpose of producing this protein in SHuffle was enhancing the capability of correctly fold proteins with multiple disulfide bonds in the cytoplasm of this strain. After purification with Ni-NTA, the purified His-tagged IL-1RA was digested with TEV protease to remove His-tag. The purpose of removing His-tag was reaching the pharmaceutical quality of IL-1RA protein in order to compare with a bioactive commercial IL-1RA protein. This study might provide a platform to produce IL-1RA protein in commercial scale in the future
- Methods: In this study, the recombinant vector named pET15b/IL-1RA/TrxA was successfully constructed and transformed into SHuffle T7 strain. The soluble form of IL-1RA/His-tag was produced in SHuffle T7 strain E. coli and purified with Ni-NTA.
Following-purification of His-tagged IL-1RA using imidazole, the His-tag was digested by TEV protease and removed. Imidazole is a potent inhibitor of TEV cleavage activity, so that the sample was first dialyzed with 5% glycerol to remove 250 mM imidazole. Then it was digested with TEV protease in a reaction buffer including 50 mM Tris-HCl (pH 8.0), 0.5 mM EDTA, and 1 mM DTT at 30° C and for 5 h. In order to improve digested pure IL-1RA, the cleaved His-tags were removed using Ni-NTA. On the other hand, EDTA as a main component of TEV reaction buffer can chelate nickel ions in the affinity gel. Thus, the digested sample was dialyzed for the second time with 5% glycerol solution before purification with Ni-NTA. After that, the digested IL-1RA was separated in the flow-through during purification, and the success of this process was evaluated using SDS-PAGE.
- Results: Based on our results, the purified IL-1RA/His-tag indicated a band corresponding to the molecular weight (Mw) of approximately 18.8 kDa. As it was shown in the lane 3, after cleavage of His-tag using TEV protease, IL-1RA protein with Mw of 17 kDa, was observed with apparent difference compared to IL-1RA/His-Tag with Mw of 18.8 kDa. After dialysis of IL-1RA to remove EDTA in TEV reaction buffer, the target protein was successfully recovered and the protein lost during dialysis steps was not significant (Lane 1, 2 and 4). The dialyzed IL-1RA was re-purified with Ni-NTA and was observed in the flow-through due to the elimination of His-tag (with Mw 17 kDa), whereas the remaining undigested IL-1RA/His-Tag was detected in the elution.
- Conclusion: In this study, for the first time IL-1RA/His-Tag was produced in soluble form in Shuffle T7 strain E. coli and His-Tag was successfully eliminated via TEV protease activity. The yield of protein production was highly determined as 9.3 mg/L. The results of this study could pave the way for large scale production of IL-1RA for therapeutic applications in the future.
- Keywords: Human IL-1RA, Recombinant protein, Shuffle T7 express E. coli, Ni-NTA, TEV protease.
