Recombinant mouse tumor necrosis factor expressed in mammalian cells: effect of glycosylation on cytotoxic activity.
A mouse tumor necrosis factor-alpha (TNF) expression vector, pTNFNeo, was constructed by inserting a 1.3 kb cDNA coding for a full structural region of mouse TNF into an expression plasmid BCMGSNeo. COS7 cells were transfected with the pTNFNeo and a G418-resistant transfectant, BK-2, which stably secreted lytic activity to L929 cells was cloned. The lytic activity in the BK-2 culture spent medium reached up to 6000 U/ml, and was completely and specifically inhibited with antiserum to mouse TNF. Gel filtration chromatography and Western blot analysis indicated that the recombinant TNF in the medium existed in associated forms composed of a mixture of 22 kDa and 17.5 kDa components. Glycopeptidase F digestion indicated that the 22 kDa species was an N-glycosylated form of the 17.5 kDa species. Specific activities of the 22 kDa and the 17.5 kDa species isolated were 6.9 x 10(5) U/mg and 8.1 x 10(6) U/mg, respectively, suggesting that carbohydrate moiety impaired the lytic activity.
Koyama
Y
Y
Tokyo Institute for Immunopharmacology, Japan.
Hayashi
T
T
Fujii
N
N
Yoshida
T
T
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D003001:Cloning, Molecular; D003601:Cytotoxicity Tests, Immunologic; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D051379:Mice; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D014170:Transformation, Genetic; D014409:Tumor Necrosis Factor-alpha / Q000737:chemistry / Q000235:genetics* / Q000276:immunology
D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha