| Autor: |
Bedanokova, D. R., Goncharuk, M. V., Shabalkina, A. V., Lushpa, V. A., Arseniev, A. S., Bocharov, E. V., Mineev, K. S., Goncharuk, S. A. |
| Zdroj: |
Russian Journal of Bioorganic Chemistry; Dec2024, Vol. 50 Issue 6, p2589-2595, 7p |
| Abstrakt: |
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. [ABSTRACT FROM AUTHOR] |
| Databáze: |
Complementary Index |
| Externí odkaz: |
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