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Biotechnology and Applied Biochemistry (2003) 37, (109–113) (Printed in Great Britain)

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Construction of a new tumour necrosis factor fusion-protein expression vector for high-level expression of heterologous genes in Escherichia coli

Wei Han, Yingqi Zhang¹, Zhen Yan and Jihong Shi

Biotechnology Center of The Fourth Military Medical University, Chang Le Xi Lu 17, Xi'an, Shaanxi 710032, People's Republic of China

Key words: affinity purification, histidine tag, P_L promoter, P_R promoter.

Abbreviations used: TNF, tumour necrosis factor; IFN, interferon; IL-11, interleukin 11; CSF, colony-forming factor; OPG, osteoprotegrin; IL-2, interleukin 2; Ni-NTA, Ni²⁺-nitrilotriacetate.

¹To whom correspondence should be addressed (e-mail Zhangyqo@fmmu.edu.cn).

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-TNFa antibody or by specific antibodies and purified by Ni²⁺-nitrilotriacetate beads. The expressed TNF fusion proteins can be cleaved by hydroxylamine.

Received 13 August 2002/13 September 2002; accepted 16 October 2002

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