Chemical synthesis, expression and product assessment of a gene coding for biologically active human tumour necrosis factor α

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Abstract
A gene encoding human tumor 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 tumor 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.

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