Binding of human tumor necrosis factor alpha to multimeric complementary peptides.
A peptide with binding properties for tumor necrosis factor (TNF alpha) sequence 144-157 has been designed, using a computer-assisted method able to create peptide sequences hydropathically complementary to a given sequence. The complementary peptide was synthesized in a multimeric form starting from an octadentate polylysine core, to facilitate its immobilization and to provide interaction multivalency. Once immobilized on a solid support to prepare an affinity column, it recognized the target TNF144-157 peptide selectively from crude peptide mixtures containing TNF fragments encompassing the entire TNF alpha sequence. Similar selectivity and specificity were shown for full-length recombinant TNF alpha, allowing its purification from crude Escherichia coli extracts. The octameric complementary peptide preserved its recognition properties for TNF alpha and biotinylated TNF alpha even after coating on microtiter plates. Competitive binding occurred with unlabeled TNF alpha in the range between 0.01 and 10 micrograms/ml, in the presence of detergent such as 0.05% Tween 20 and in the presence of 1% normal goat serum. The effect of complementary peptide multimerization was evidenced by its enhanced binding affinity for TNF alpha, which exists in solution as a trimer, while the target TNF[144-157] peptide was recognized with much lower strength. The dissociation constant for interaction with TNF alpha was close to 10 nM, allowing its easy detection by solid phase assays in concentrations as low as 10 pmol/ml.
Fassina
G
G
Protein Engineering Unit, TECNOGEN S.c.p.A., Milan, Italy.
Cassani
G
G
Corti
A
A
D000595:Amino Acid Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D003201:Computers; D015195:Drug Design; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D011487:Protein Conformation; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
D009842:Oligopeptides; D010446:Peptide Fragments; D014409:Tumor Necrosis Factor-alpha