|تعداد مشاهده مقاله||10,670,283|
|تعداد دریافت فایل اصل مقاله||5,133,168|
Introducing a New Method for Purification of Human IL-4 by Substitution of a Single Amino Acid in IL-4 Protein Sequence
|Iranian Journal of Immunology|
|دوره 19، شماره 4، اسفند 2022، صفحه 436-445 اصل مقاله (2.46 M)|
|نوع مقاله: Original Article|
|شناسه دیجیتال (DOI): 10.22034/iji.2022.94985.2336|
|Mohsen Mazloomrezaei 1؛ Mahsa Sadat Hosseini 1؛ Nahid Ahmadi1، 2؛ Elham Mahmoudi Maymand 1؛ Ebrahim Eftekhar 3؛ Amir Asgari 4؛ Amin Ramezani 1، 2|
|1Shiraz Institute for Cancer Research, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran|
|2Department of Medical Biotechnology, School of Advanced Medical Sciences and Technologies, Shiraz University of Medical Sciences, Shiraz, Iran|
|3Molecular Medicine Research Center, Hormozgan Health Institute, Hormozgan University of Medical Sciences, Bandar Abbas, Iran|
|4Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, Canada|
| 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.
|Cyanogen Bromide؛ Interleukin-4؛ Protein Purification؛ Tag removal|
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