The limited proteolysis of tumor necrosis factor-alpha.
The limited proteolysis of human recombinant TNF-alpha by trypsin yields two stable products resulting from cleavage after Arg6 and Arg44. In solution these two products remain associated together in a trimer with a Stokes' radius slightly greater than the radius of intact TNF-alpha and, therefore, could not be separated from each other under nondenaturing conditions. This limited digest retains at least 20% of the activity of the original TNF-alpha sample, and has a tertiary structure that is similar to that of the native protein by circular dichroism. On the other hand, incorrectly folded, inactive TNF-alpha undergoes extensive digestion following similar treatment with trypsin. These results indicate that the active form of TNF-alpha has a tight core structure which is maintained after N-terminal cleavage and removal.
Narhi
L O
LO
Amgen Inc., Thousand Oaks, California 91320.
Rhode
M F
MF
Hunt
P
P
Arakawa
T
T
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000302:isolation & purification; D011487:Protein Conformation; D012697:Serine Endopeptidases; D014357:Trypsin; D014409:Tumor Necrosis Factor-alpha / Q000302:isolation & purification*
D010446:Peptide Fragments; D014409:Tumor Necrosis Factor-alpha; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D014357:Trypsin