[Purification of recombinant proteins with an example of tumor necrosis factor thymosin-alpha1].
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.
Fedorov
T V
TV
Korobov
V I
VI
Nazarov
V G
VG
Smolkina
A E
AE
Shmelev
V A
VA
D000818:Animals; D045744:Cell Line, Tumor; D002850:Chromatography, Gel / Q000379:methods*; D004229:Dithiothreitol / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D002479:Inclusion Bodies / Q000737:chemistry; D051379:Mice; D011994:Recombinant Proteins / Q000302:isolation & purification* / Q000494:pharmacology; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D000077596:Thymalfasin; D013947:Thymosin / Q000031:analogs & derivatives* / Q000302:isolation & purification / Q000494:pharmacology; D014462:Ultrafiltration
D011994:Recombinant Proteins; D012967:Sodium Dodecyl Sulfate; D013947:Thymosin; D004229:Dithiothreitol; D000077596:Thymalfasin