1. [Purification of recombinant proteins with an example of tumor necrosis factor thymosin-alpha1].
- Author
-
Fedorov TV, Korobov VI, Nazarov VG, Smolkina AE, and Shmelev VA
- Subjects
- Animals, Cell Line, Tumor, Dithiothreitol chemistry, Escherichia coli genetics, Escherichia coli metabolism, Inclusion Bodies chemistry, Mice, Recombinant Proteins pharmacology, Sodium Dodecyl Sulfate chemistry, Thymalfasin, Thymosin isolation & purification, Thymosin pharmacology, Ultrafiltration, Chromatography, Gel methods, Recombinant Proteins isolation & purification, Thymosin analogs & derivatives
- Abstract
Hybrid protein, cancer necrosis factor thymosin-alpha1 (CNF-T), when synthesizing in strain-producer of Escherichia coli SG200-50 with plasmid pThy315, was a part of "inclusion bodies" mostly in the form of a high-molecular complex with other proteins due to the S-S bonds formation. An approach of purification of CNF-T has been proposed, which is based on the destruction of the complex in the presence of sodium dodecylsulfate (DDS-NA) and dithiotreitol (DDT) followed by gel-filtration on Sephadex G-100 and renaturation by ultrafiltration on hollow fibers. The method allows the isolation of electrophoretically homogeneous CNF-T containing no DDS-Na and having high cytotoxic activity against cancer cells of mouse adenocarcinome L-929. The yield of CNF-T achieved 80% relative its content in biomass and 30% relative the total protein.
- Published
- 2010