Expression of a partial synthetic human TNF cDNA in E. coli.
A recombinant human tumour necrosis factor (rhTNF) cDNA was constructed. The TNF gene was isolated from a human genomic gene library. There are four exons in the TNF gene. The fourth exon codes for 140 amino acids of the TNF matured protein which is composed of 157 amino acids. A major portion of the fourth exon was isolated and then ligated to a synthesized DNA fragment coding for the remaining amino acids. The partial synthetic hTNF (rhTNF) cDNA thus generated was subcloned into a vector and successfully expressed in E. coli. 5-1 fermentator was used to produce rhTNF. About 20 g (wet weight) of bacterial pellet per liter medium and 10(6)-10(7) units of cytotoxicity to L929 cells per milliliter medium were obtained. rhTNF was purified by HPLC and dried with a freeze dryer. rhTNF with a purity of about 95% in the form of white powder was obtained. The sequence of ten amino acids at the amino terminus of the rhTNF was determined. The result showed that it was identical with that of the natural human TNF.
Li
C B
CB
State Key Laboratory of Genetic Engineering, Fudan University, Shanghai, PRC.
Chai
C X
CX
Xie
Y
Y
Lü
Q
Q
Chai
J H
JH
Yu
H
H
Chen
C M
CM
Lin
L Z
LZ
Wang
Q S
QS
Li
Z L
ZL
D001483:Base Sequence; D003001:Cloning, Molecular; D004274:DNA, Recombinant / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D005091:Exons; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis* / Q000235:genetics*
D004274:DNA, Recombinant; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha