Purification of recombinant human tumor necrosis factor precursor from Escherichia coli.
To study its biological functions, tumor necrosis factor precursor (proTNF) with a molecular size of 26-KDa was obtained as a recombinant protein from Escherichia coli. The recombinant proTNF was successfully accumulated in the insoluble form, corresponding to about 10-15% of total E. coli proteins. Solubilization, gel filtration and anion exchange chromatography were performed under denatured conditions followed by dialysis in phosphate-buffered saline. These processes removed most of the contaminating bacterial proteins, yielding proTNF with a purity of about 70-80%. This recombinant proTNF is expected to be useful for functional studies on activated macrophages with membrane integrated proTNF.
Tanabe
Y
Y
Biotechnology Research Center, Teikyo University, Kawasaki 2, Japan.
Noguchi
K
K
Morikawa
A
A
Mizuno
D
D
Soma
G
G
D015153:Blotting, Western; D002460:Cell Line; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D015723:Gene Library; D006801:Humans; D008970:Molecular Weight; D011498:Protein Precursors / Q000235:genetics / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000302:isolation & purification; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000302:isolation & purification*
D011498:Protein Precursors; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; C061153:tumor necrosis factor precursor