Construction of Expression Vectors for Efficient Production of Recombinant Proteins in E. coli for the Development of Therapeutic Drugs

IF 0.6 Q4 BIOCHEMISTRY & MOLECULAR BIOLOGY Biochemistry (Moscow), Supplement Series B: Biomedical Chemistry Pub Date : 2024-11-05 DOI:10.1134/S1990750823600516
M. V. Zakharova, E. K. Mubarakshina, M. O. Nagornykh
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Abstract

When developing vaccines and immunotherapeutic and enzymatic drugs, it is usually necessary to obtain a functional protein preparation, which can later be used for immunization or analytical studies in the process of creating a drug. Production in E. coli bacterial cells is the fastest and cheapest way to obtain such proteins. However, recombinant proteins, when produced in E. coli, often form insoluble and functionally inactive aggregates. The purpose of this work is to create a set of expression vectors for rapid screening of optimal conditions for the production of recombinant proteins prone to aggregation in E. coli in soluble form. The work used modern genetic engineering methods, including optimization and de novo synthesis of nucleotide sequences encoding helper polypeptides. For the production and chromatographic purification of proteins, the standard strain BL21(DE3) and the affinity chromatography method using a metal chelate sorbent were used. As a result, a set of nine vectors with helper polypeptides was obtained to increase solubility and increase product yield during the production of recombinant proteins in E. coli cells. The efficiency of obtaining protein drugs prone to molecular aggregation using this set of vectors has been demonstrated by the exampe of three cytokines: interleukin-31 (IL-31), interleukin-33 (IL-33), and transforming growth factor beta (TGF-beta1). The resulting set allows for rapid selection of a suitable helper polypeptide as well as optimization of conditions for obtaining a soluble form of recombinant proteins in E. coli during the pharmaceutical development of therapeutic drugs.

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构建表达载体,在大肠杆菌中高效生产重组蛋白以开发治疗药物
在开发疫苗、免疫治疗药物和酶制剂时,通常需要获得一种功能性蛋白质制剂,这种制剂以后可用于免疫接种或药物生产过程中的分析研究。在大肠杆菌细胞中生产是获得这种蛋白质的最快、最便宜的方法。然而,在大肠杆菌中生产的重组蛋白往往会形成不溶性和功能不活跃的聚集体。这项工作的目的是创建一套表达载体,用于快速筛选在大肠杆菌中生产易聚集的可溶性重组蛋白的最佳条件。这项工作采用了现代基因工程方法,包括优化和从头合成编码辅助多肽的核苷酸序列。在蛋白质的生产和色谱纯化过程中,使用了标准菌株 BL21(DE3)和使用金属螯合吸附剂的亲和层析方法。结果,获得了一套含有辅助多肽的九种载体,可在大肠杆菌细胞中生产重组蛋白时增加溶解度和产品产量。白细胞介素-31(IL-31)、白细胞介素-33(IL-33)和转化生长因子 beta(TGF-beta1)这三种细胞因子的试验证明,使用这组载体可以有效地获得容易发生分子聚集的蛋白质药物。利用这套载体可以快速选择合适的辅助多肽,并优化条件,以便在治疗药物的制药开发过程中在大肠杆菌中获得可溶性重组蛋白。
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来源期刊
CiteScore
1.10
自引率
0.00%
发文量
31
期刊介绍: Biochemistry (Moscow), Supplement Series B: Biomedical Chemistry   covers all major aspects of biomedical chemistry and related areas, including proteomics and molecular biology of (patho)physiological processes, biochemistry, neurochemistry, immunochemistry and clinical chemistry, bioinformatics, gene therapy, drug design and delivery, biochemical pharmacology, introduction and advertisement of new (biochemical) methods into experimental and clinical medicine. The journal also publishes review articles. All issues of the journal usually contain solicited reviews.
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