Downstream technique study of human tumor necrosis factor alpha and its mutant.
The study of downstream techniques for recombinant human necrosis factor alpha (rhTNF alpha) and its mutant (Lys2)-rhTNF alpha led to the results of approximately 50 g wet recombinant E. coli per liter with high expression level (> 50%) harvested from autocontrolled fed-batch culture in 15 L fermentor (B. Braun). The rhTNF alpha and (Lys2)-rhTNF alpha expressed are totally soluble. Followed by the process of ultrasonication, ammonium sulfate precipitation, ion-exchange chromatography (DEAE-sepharose FF, CM-sepharose FF), and molecular sieve (Sephacryl S-200), a yield of approximately 1 g pure recombinant protein from 1 L broth is obtained. The purity is up to 98%. The specific activity of rhTNF alpha and (Lys2)-rhTNF alpha are approximately 1.5 x 10(8) IU/mg and approximately 6 x 10(8) IU/mg, respectively.
Xu
H
H
Shanghai Research Center of Biotechnology, Chinese Academy of Sciences, China.
Gao
C
C
Liu
H
H
Deng
F
F
Pan
H
H
Chen
C
C
D004926:Escherichia coli; D005285:Fermentation; D006801:Humans; D008239:Lysine / Q000235:genetics; D016296:Mutagenesis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; D008239:Lysine