Characterization of the 14 kda fragment of human tumor-necrosis-factor-alpha |
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Authors: | Sagoo J Iaonnou C Beeley N Sutton C Dematteis C Tendler S |
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Institution: | UNIV NOTTINGHAM,DEPT PHARMACEUT SCI,BIOPHYS & SURFACE ANAL LAB,NOTTINGHAM NG7 2RD,ENGLAND. CELLTECH LTD,SLOUGH SL1 4EN,BERKS,ENGLAND. FINNIGAN MAT CORP,HEMEL HEMPSTEAD HP2 4TG,HERTS,ENGLAND. |
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Abstract: | 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. |
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