Construction and Expression of hTNFalpha-hTGF3 Fused Gene.
Human tumor necrosis factor was fused with hTGF3, the third loop region of hTGFalpha specifically binding to EGF receptor, through a flexible linker by recombinant DNA techniques. The constructed plasmid containing hTNFalpha-hTGF3 fused gene, pSB92-TLT, was expressed under the control of P(L) promoter and after the induction at 42 degrees it gave a high level of expression of the fused gene, ranging in 50%-60% of total bacterial protein. 95% of the expressed product was inclusion body, and only 5% was soluble. When the fused gene was constructed into the constitutive plasmid pLC, a new plasmid controlled by P(L) promoter but without cIts857, and expressed at 30 degrees, the expression yield reached 50% and a high proportion of soluble protein, 35% of total bacterial protein, was obtained. Western blot analysis indicated that the fusion protein could specifically bind to anti-hTNFalpha antibody. Comparing to original hTNFalpha, the fused hTNFalpha derivative showed about equal cytotoxicity to L929 cell line and higher cytotoxicity to some human tumor cell lines. This shows that the fusion protein TLT may be promising in application.
Shanghai Research Center of Biotechnology, the Chinese Academy of Sciences, Shanghai 200233, China. cqchen@server.shcnc.ac.cn
Jian-Jun
JJ
Chen
Miao
M
Sun
Fang
F
Lu
Chang-Qing
CQ
Chen
De-Bao
DB
Wang