High expression of tumor necrosis factor alpha cDNA in E. coli using site-specific deletion mutagenesis.
Using oligonucleotide-directed site specific mutagenesis technique, a TNF-alpha cDNA coding 157 amino acids without signal sequence and initiated with ATG was obtained. Sequencing data showed the precise deletion and insertion of the DNA fragment as designed. The modified TNF gene was expressed in E. coli with a expression vector pBV220. The expression level reached 10(8) units per liter culture. The scanning of the SDS-PAGE gel showed that the amount of the expressed TNF is about 22.8% of total soluble bacterial protein. The TNF activity can be neutralized with TNF monoclonal antibody.
Zhang
D Z
DZ
National Laboratory of Molecular Virology and Genetic Engineering, Beijing.
Wu
S H
SH
Zhang
Z Q
ZQ
Jin
Q
Q
Zhao
X H
XH
Zhao
X X
XX
Su
C Z
CZ
Hou
Y D
YD
D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009500:Neutralization Tests; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics*
D014409:Tumor Necrosis Factor-alpha; D004247:DNA