Construction of a new tumour necrosis factor fusion-protein expression vector for high-level expression of heterologous genes in Escherichia coli.
We report the construction and application of a new fusion-protein expression plasmid (TNFHis) for Escherichia coli. The plasmid contains both P(R) and P(L) promoters and is optimized to allow a higher level of expression of mature coding sequences. It also contains a six-histidine tag for convenient purification as well as thrombin and hydroxylamine recognition sites for cleaving heterologous protein. The potential use of this expression vector is demonstrated by comparing the expression levels of human tumour necrosis factor (TNF), interferon, interleukin 11, colony-forming factor, osteoprotegrin and interleukin 2 in E. coli. Furthermore, all expressed TNF fusion proteins can be detected by anti-TNF alpha antibody or by specific antibodies and purified by Ni(2+)-nitrilotriacetate beads. The expressed TNF fusion proteins can be cleaved by hydroxylamine.
Han
Wei
W
Biotechnology Center of The Fourth Military Medical University, Chang Le Xi Lu #17, Xi'an, Shaanxi 710032, People's Republic of China.
Zhang
Yingqi
Y
Yan
Zhen
Z
Shi
Jihong
J
D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics*; D005822:Genetic Vectors; D006023:Glycoproteins / Q000378:metabolism; D006801:Humans; D007372:Interferons / Q000378:metabolism; D007376:Interleukin-2 / Q000378:metabolism; D053244:Osteoprotegerin; D010957:Plasmids / Q000235:genetics*; D011401:Promoter Regions, Genetic; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D018124:Receptors, Tumor Necrosis Factor; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification
D006023:Glycoproteins; D007376:Interleukin-2; D053244:Osteoprotegerin; D018160:Receptors, Cytoplasmic and Nuclear; D018124:Receptors, Tumor Necrosis Factor; D011993:Recombinant Fusion Proteins; C506030:TNFRSF11B protein, human; D014409:Tumor Necrosis Factor-alpha; D007372:Interferons