Rekombinantinio TDP-43 baltymo sintezės ir gryninimo optimizavimas /

The aim of this work is to optimize the synthesis and purification of recombinant SUMO-TDP-43 protein in E. coli cells. The targeting protein is important in the context of neurodegenerative diseases, as accumulations of amyloid TDP-43 aggregates cause cell damage. The preparations carried out in this work included the synthesis of the SUMO-TDP-43 construct and purification optimization work. E.coli BL21 (DE3), Rosetta 2 (DE3), Star (DE3), Gold (DE3)) strains were transformed with SUMO-TDP-43 protein expression vectors and an attempt was made to select the optimal one. E.coli BL21 Star (DE3) and Gold (DE3) transformation efficiency and growth rate were higher than other strains. Based on the results, work were continued with the E. coli BL21 Star (DE3) strain. The importance of the choice of nutrient medium for protein solubility was also evaluated. Among Luria-Bertani (LB), autoinductive ZYM-5052 and minimal M9 medium, autoinductive ZYM-5052 medium was chosen as the most favorable due to its high final biomass content. A buffer solution with sarkosyl was not effective in maintaining the solubility of the target protein, possibly due to denaturation or aggregation of the protein, and a solution of 5% glycerol and Triton X-114 was also not effective in maintaining the protein in the soluble fraction. A buffer solution with 10% glycerol was the most effective as it provided the highest solubility of the target protein. The effect of pH on the solubility of SUMO-TDP-43 protein in the 5-8 range was also evaluated. At pH 5, the SUMO-TDP-43 protein became unstable and insoluble, likely due to misfolding or denaturation. As the pH increased, the results improved, and the highest tested pH (pH 8) was optimal for protein stability and solubility without precipitation. The results of the studies showed that the solubility and stability of the protein can be optimized depending on the choice of strain, composition of buffer solutions, imidazole concentrations gradient during IMAC and the appropriate pH level. Control of these factors is essential for efficient extraction and purification of target proteins in recombinant protein assays. The data and conclusions of this work may help to develop a stable protocol for SUMO-TDP-43 protein synthesis.

Grantor dc:publisher

Institutional Repository of Vilnius University

Year dc:date

2024

Author dc:creator

• Dovidovičiūtė, Gabija,

Statement dc:rights

• info:eu-repo/semantics/openAccess

Language dc:language

lit

Repository record dc:identifier

https://repository.vu.lt/VU:ELABAETD210638762&prefLang=en_US

OAI identifier oai:identifier

oai:vu.lt:elaba:210638762