The role of amino functions in recombinant human tumor necrosis factor in expression of biological activity.
The role of amino functions in the expression of the biological activity of recombinant human TNF (rHuTNF) was studied by chemical modification. rHuTNF is a homotrimer of 17 kD subunits, each of which contains an N-terminal valine and six lysyl residues: two of these lysyl residues are known to be involved in intra- or intersubunit interactions. Chemically reactive amino functions were modified with the N-hydroxysuccinimide ester of acetic acid; modification of amino groups to amide, and the concomitant loss of charge, was monitored by native PAGE. When rHuTNF was reacted with the active ester at increasing mole ratios, up to 12 amino groups per trimer could be modified. When the biological activity of acetylated rHuTNF was determined, a strong correlation between the extent of modification and loss of biological activity was observed. One to three amino groups per trimer could be modified with nearly complete retention (approximately 80-95%) of biological activity; activity was essentially completely destroyed at the highest levels of modification. These results reveal important functions for the amino groups of rHuTNF and significant constraints on strategies involving their modification in development of second-generation-TNF variants.
Utsumi
T
T
Department of Tumor Biology, University of Texas, M. D. Anderson Cancer Center, Houston 77030.
Hung
M C
MC
Klostergaard
J
J
D000107:Acetylation; D000588:Amines / Q000737:chemistry; D016923:Cell Death; D006801:Humans; D066298:In Vitro Techniques; D011994:Recombinant Proteins / Q000737:chemistry; D013329:Structure-Activity Relationship; D014409:Tumor Necrosis Factor-alpha / Q000737:chemistry* / Q000633:toxicity
D000588:Amines; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha