M. V. Zakharova, E. K. Mubarakshina, M. O. Nagornykh
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引用次数: 0
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.
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.