通过铜催化空气氧化进行氧化重折叠可持续提高新型白细胞介素-2静音素的均一性和活性。

IF 4.1 2区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Journal of biotechnology Pub Date : 2024-07-18 DOI:10.1016/j.jbiotec.2024.07.013
Sum Lai Lozada , Jose Alberto Gómez , Katherine Menéndez , Tania Gómez , Daidee Montes de Oca , Jose L. Durán , Olga Lidia Fernández , Yoel Perera , Gabriela Rivas , Tammy Boggiano-Ayo , Nuris Ledon , Tania Carmenate
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引用次数: 0

摘要

白细胞介素-2(IL-2)用于癌症治疗已有 30 多年的历史。然而,由于其毒性较高,人们开发出了新的突变变体。这些变体保留了原始分子的部分生物特性,但具有其他治疗优势。在分子免疫学中心,IL-2 no-α mutein(一种毒性低于 wtIL-2 的 IL-2 激动剂)已经设计、生产出来,目前正在进行 I/II 期临床试验评估。静蛋白是在大肠杆菌中生产的不溶性物质,必须在体外重新折叠才能产生完全活性的蛋白质。在大肠杆菌中生产的重组蛋白的纯化过程中,受控氧化步骤至关重要,以确保分子中二硫键的正常形成。在这种情况下,新的纯化过程包括铜催化的空气氧化步骤,以诱导二硫键的形成。通过筛选,确定了这一步骤的最佳 pH 值、铜、蛋白质和洗涤剂浓度条件。与不经过氧化步骤的现有工艺相比,生产出的蛋白质具有稳定的三维结构、更高的纯度和更强的生物活性。生产并评估了四批产品,证明了新工艺的一致性。
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Oxidative refolding by Copper-catalyzed air oxidation consistently increases the homogeneity and activity of a Novel Interleukin-2 mutein

Interleukin-2 (IL-2) has been used in cancer treatment for over 30 years. However, due to its high toxicity, new mutant variants have been developed. These variants retain some of the biological properties of the original molecule but offer other therapeutic advantages. At the Center of Molecular Immunology, the IL-2 no-alpha mutein, an IL-2 agonist with lower toxicity than wtIL-2, has been designed, produced, and is currently being evaluated in a Phase I/II clinical trial. The mutein is produced in E. coli as an insoluble material that must be refolded in vitro to yield a fully active protein. Controlled oxidation steps are essential in the purification process of recombinant proteins produced in E. coli to ensure the proper formation of the disulfide bonds in the molecules. In this case, the new purification process includes a copper-catalyzed air oxidation step to induce disulfide bond establishment. The optimal conditions of pH, copper, protein and detergent concentration for this step were determined through screening. The produced protein demonstrated a conserved 3D structure, higher purity, and greater biological activity than the obtained by established process without the oxidation step. Four batches were produced and evaluated, demonstrating the consistency of the new process.

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来源期刊
Journal of biotechnology
Journal of biotechnology 工程技术-生物工程与应用微生物
CiteScore
8.90
自引率
2.40%
发文量
190
审稿时长
45 days
期刊介绍: The Journal of Biotechnology has an open access mirror journal, the Journal of Biotechnology: X, sharing the same aims and scope, editorial team, submission system and rigorous peer review. The Journal provides a medium for the rapid publication of both full-length articles and short communications on novel and innovative aspects of biotechnology. The Journal will accept papers ranging from genetic or molecular biological positions to those covering biochemical, chemical or bioprocess engineering aspects as well as computer application of new software concepts, provided that in each case the material is directly relevant to biotechnological systems. Papers presenting information of a multidisciplinary nature that would not be suitable for publication in a journal devoted to a single discipline, are particularly welcome.
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