Document Type : Original Article
Authors
- Mohsen Mazloomrezaei 1
- Mahsa Sadat Hosseini 1
- Nahid Ahmadi 1, 2
- Elham Mahmoudi Maymand 1
- Ebrahim Eftekhar 3
- Amir Asgari 4
- Amin Ramezani 1, 2
1 Shiraz Institute for Cancer Research, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran
2 Department of Medical Biotechnology, School of Advanced Medical Sciences and Technologies, Shiraz University of Medical Sciences, Shiraz, Iran
3 Molecular Medicine Research Center, Hormozgan Health Institute, Hormozgan University of Medical Sciences, Bandar Abbas, Iran
4 Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, Canada
Abstract
Background: It is advantageous to develop an effective purification procedure to produce recombinant protein drugs (rPDs) without any tags. To remove N- or C-terminus tags from the rPDs, several cleavage site-based endopeptidases were used. Separating the endopeptidase enzyme from the rPDs is a time-consuming and costly process.
Objective: To design and develop a new method for the purification of human interleukin (IL)-4 with potential application for other cytokines.
Methods: Met-like amino acids were substituted at position 120 to reduce the possibility of alteration in the structure of IL-4 and its biological activity. Based on the in silico analysis, isoleucine was chosen as an alternative amino acid, and the M120I mutant IL-4 (mIL-4) model was selected for the downstream analysis. Recombinant mIL-4 was produced in the E.coli BL21 host and purified with CNBr. Then in vitro evaluations of the native and mutant IL-4 were performed.
Results: The results showed that both the native and mutant IL-4 had the same effect on TF-1 cell proliferation. On the other hand, there was no significant difference between the effects of native IL-4 (nIL-4) and mIL-4 on the expression of IL-4 and IL-10 in activated peripheral blood mononuclear cells. Native and mutant IL-4 have similar biological activities.
Conclusion: Here, an efficient and straightforward system is introduced to purify IL-4 cytokine using CNBr, which could be applied to other rPDs.
Keywords
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