Introducing a New Method for Purification of Human IL-4 by Substitution of a Single Amino Acid in IL-4 Protein Sequence.

IF 1.1 4区医学 Q4 IMMUNOLOGY

Iranian Journal of Immunology Pub Date : 2022-12-01 DOI:10.22034/IJI.2022.94985.2336

Mohsen Mazloomrezaei, Mahsa Sadat Hosseini, Nahid Ahmadi, Elham Mahmoudi Maymand, Ebrahim Eftekhar, Amir Asgari, Amin Ramezani

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引用次数: 0

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.

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介绍了一种通过替换IL-4蛋白序列中单个氨基酸纯化人IL-4的新方法。

背景:开发一种有效的纯化方法来生产不带任何标签的重组蛋白药物(rpd)是非常有利的。为了从rpd中去除N或c端标签，使用了几种基于切割位点的内多肽酶。从rpd中分离内肽酶是一个耗时且昂贵的过程。目的:设计并建立一种纯化人白细胞介素-4的新方法，该方法具有纯化其他细胞因子的潜力。方法:在120位取代类met氨基酸，降低IL-4结构改变的可能性，降低其生物活性。在硅分析的基础上，选择异亮氨酸作为替代氨基酸，并选择M120I突变体IL-4 (mIL-4)模型进行下游分析。在大肠杆菌BL21宿主中产生重组mIL-4，并用CNBr纯化。然后对原生和突变IL-4进行体外评价。结果:结果表明，天然IL-4和突变IL-4对TF-1细胞增殖的影响相同。另一方面，天然IL-4 (nIL-4)和mIL-4对活化的外周血单个核细胞中IL-4和IL-10表达的影响无显著差异。原生和突变的IL-4具有相似的生物活性。结论:本研究建立了一种高效、简便的CNBr纯化IL-4细胞因子系统，该系统可应用于其他rpd。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Iranian Journal of Immunology Medicine-Immunology and Allergy

CiteScore

1.60

自引率

0.00%

发文量

审稿时长

12 weeks

期刊介绍： The Iranian Journal of Immunology (I.J.I) is an internationally disseminated peer-reviewed publication and publishes a broad range of experimental and theoretical studies concerned with all aspects of immunology.