TrkA 受体配体结合域与胞外并膜区的制作和重构

IF 1.1 4区 化学 Q4 BIOCHEMISTRY & MOLECULAR BIOLOGY
D. R. Bedanokova, M. V. Goncharuk, A. V. Shabalkina, V. A. Lushpa, A. S. Arseniev, E. V. Bocharov, K. S. Mineev, S. A. Goncharuk
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引用次数: 0

摘要

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Production and Refolding of the Ligand-Binding Domain of TrkA Receptor with the Extracellular Juxtamembrane Region

Production and Refolding of the Ligand-Binding Domain of TrkA Receptor with the Extracellular Juxtamembrane Region

Objective: One of the major problems in the field of neurotrophin signaling is the role of Trk juxtamembrane regions. Here we present the production protocol of the d5 ligand-binding domain of TrkA with the full-length extracellular juxtamembrane region for structural studies. Methods: The protein was produced in E. coli cells. Protein purification included immobilized metal affinity and size-exclusion chromatography in the presence of urea. Refolding was performed using three approaches: dialysis, pulse and flash dilution. The quality of the final protein was assessed by gel filtration and NMR. Results and Discussion: We demonstrated that the obtained strain allows the production of milligram quantities of the target protein, including its isotope-labeled derivatives. A comparison of several refolding protocols revealed that dialysis and flash dilution are optimal, with the latter option being more economically feasible. Conclusions: Analysis of the final protein preparation showed that the proposed protein expression, purification, and refolding scheme allows the production of a highly purified protein suitable for structural and functional studies.

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来源期刊
Russian Journal of Bioorganic Chemistry
Russian Journal of Bioorganic Chemistry 生物-生化与分子生物学
CiteScore
1.80
自引率
10.00%
发文量
118
审稿时长
3 months
期刊介绍: Russian Journal of Bioorganic Chemistry publishes reviews and original experimental and theoretical studies on the structure, function, structure–activity relationships, and synthesis of biopolymers, such as proteins, nucleic acids, polysaccharides, mixed biopolymers, and their complexes, and low-molecular-weight biologically active compounds (peptides, sugars, lipids, antibiotics, etc.). The journal also covers selected aspects of neuro- and immunochemistry, biotechnology, and ecology.
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