Overexpression of a Novel Highly Active Tumor Necrosis Factor Mutant in E. coli.
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.
Gao
Chang-Shou
CS
Shanghai Research Centre of Biotechnology, Academia Sinica, Shanghai 200233, China.
Mao
Shen-Lan
SL
Yu
Ying
Y
Chen
Chang-Qing
CQ