title: Overexpression of a Novel Highly Active Tumor Necrosis Factor Mutant in E. coli.
abstract: A synthetic gene of human tumor necrosis factor alpha (hTNF-alpha) was mutated by polymerase chain reaction using a multi-site mutated primer with the Arg-2 codon (CGT) replaced by a lysine codon (AAA). The mutated gene was inserted into vector pSB-92 at the downstream of P(L) promoter to give a recombinant plasmid pBS-TNF-K2 and expressed in E. coli. Soluble [Lys(2)] hTNF-alpha mutein was obtained in high yield, accounting for more than 60% of the total cellular protein and easy to purify. Compared to the wild type hTNF-alpha, purified [Lys(2)] hTNF-alpha has slightly less toxicity but shows tens to hundreds times greater inhibitory activity to several human tumor cell lines.
authors.affiliation: Shanghai Research Centre of Biotechnology, Academia Sinica, Shanghai 200233, China.
authors.forename: Chang-Shou
authors.initials: CS
authors.lastname: Gao
authors.forename: Shen-Lan
authors.initials: SL
authors.lastname: Mao
authors.forename: Ying
authors.initials: Y
authors.lastname: Yu
authors.forename: Chang-Qing
authors.initials: CQ
authors.lastname: Chen