Characterization of the 14 kda fragment of human tumor-necrosis-factor-alpha

We report the characterization of a 14 kDa degradation fragment from recombinant human tumour necrosis factor-alpha (TNF alpha) by N-terminal sequencing and mass spectrometry. A single site between the dibasic residues Arg(31)-Arg(32) of the mature recombinant 17 kDa protein has been identified as the target site that generates the 14 kDa fragment. The observation that a maximum of 33% degradation occurs suggests that only one monomer per TNF trimer is cleaved. E. coli proteases specific for dibasic residues are thought to be responsible for this cleavage. A strategy has been developed which completely inhibits proteolysis. This strategy has been used to reduce the 14 kDa degradation fragment obtained from approximately 33% of the total purified protein to zero.