Chemical synthesis, expression and product assessment of a gene coding for biologically active human tumour necrosis factor alpha.
A gene encoding human tumour necrosis factor alpha (TNF-alpha) has been chemically synthesized, cloned and expressed to yield a biologically active protein in Escherichia coli. The 480-bp gene was assembled by enzymic ligation of 32 oligonucleotides, cloned directly into M13mp18 for sequence verification and expressed in the broad host range high-level expression vector pMMB66EHST. Expressed recombinant TNF-alpha was shown to have the correct molecular weight, processed N-terminal sequence, antibody cross-reactivity and tumour cell killing activity. The expression product of the synthetic gene has been purified to homogeneity by a two-step ion-exchange procedure and the purified material shown to be active.
Ashman
K
K
European Molecular Biology Laboratory, Heidelberg, FRG.
Matthews
N
N
Frank
R W
RW
D000595:Amino Acid Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics*
D014409:Tumor Necrosis Factor-alpha