Author/Editor		Menart, V; Gaberc-Porekar, V; Kraševec, N; Miličić, S; Komel, R
Title		Production of recombinant human tumor necrosis factor alpha in two different Escherichia coli expression systems
Translated title		Produkcija rekombinantnega humanega faktorja tumorske nekroze alfa z dvema ekspresijskima sistemoma v bakteriji Escherichia coli
Type		članek
Source		Zdrav Vestn
Vol. and No.		Letnik 63, št. Suppl 2
Publication year		1994
Volume		str. II-43-II-4
Language		eng
Abstract		Background. For biosynthesis of many recombinant proteins Escherichia coli (E. coli) is a very suitable host organism due to easy manipulation and availability of data concerning its genetics, plasmids etc. We explored the applicability of expression plasmids pMAX and pCYTEXP1 for the laboratory production of human tumor necrosis factor alpha (TNF). Methods. For the reconstruction of plasmid and insertion of TNF gene standard methods of recombinant DNA technology were used. For isolation of the pure protein known chromatographic techniques were applied, taking into account the conditions which maintain biological activity of TNF. In assessing the quality of the purified protein, the main criteria were sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)-single band, high specific activity (cytotoxicity) and the absence of DNA and endotoxin. Results. In the system E. coli JM109/pMAX-TNF, the fusion protein adenylate kinase-TNF (ADK-TNF) is accumulated within the cell as insoluble inclusion bodies. Expression level was estimated to be about 10 to 30 per cent. Inclusion bodies were dissolved, than the ADK fragment was cleaved from TNF with CNBr. Crude mixture was separated on preparative SDS-PAGE. TNF was electroeluted from the gel and in the same time renatured in a dialysis bag. We obtained a pure protein (single band on SDS-PAGE) having specific activity of 1-2 x 10 on power 6 U/mg. Purification yield was 30-40 per cent. With the system E. coli TG1/pCYTEXP1-TNF, a stable expression of soluble, biologically active TNF was achieved at the level of 1-5 per cent of total cytoplasmic proteins. For purification we applied anionic chromatography, hydrophobic interaction chromatography (HIC) and preparative non-denaturing PAGE as the main separation steps. The final yield of TNF, having high specific activity of 2-3 x 10 on power 7 U/mg, was 10-20 per cent. The content of endotoxin, determined by Limulus Amebocyte Lysate (LAL) test, was less than 0.(trunc.
Summary		)
Descriptors		ESCHERICHIA COLI PLASMIDS TUMOR NECROSIS FACTOR RECOMBINATION, GENETIC TRANSFORMATION, GENETIC