title: Expression of synthetic human tumor necrosis factor is toxic to Escherichia coli.
abstract: The overlap forward-primer-walk polymerase chain reaction method was used to synthesize the human tumor necrosis factor α (hTNF) gene in Escherichia coli cells. Growth curves for hTNF and pET23d vector cultures exhibited slower doubling rates than cultures containing the pET23d vector alone. Cell cultures transformed with hTNF reached peak densities (0.4-0.6 OD(600)) 3 to 4 h post-induction, then decreased prior to growth recovery. This inhibition occurred in the BL21DE3 strain of E. coli, whereas no inhibition of growth and no expression of hTNF were observed in the JM109 strain of E. coli containing hTNF. Induced hTNF cultures hyperexpressed the hTNF-histidine fusion protein for the first 3 to 4h of induction; subsequently, growth retardation was observed. Hyperexpression and continuous growth were observed in the extracellular expression system. Electron microscopy revealed that accumulation of hTNF inclusion bodies was apparent only in the intracellular expression system - no accumulation was observed with regard to the secretory system. The hTNF-pET23d vector was purified from cells expressing the fusion protein and from cells with recovered growth curves. Sequencing of the vector demonstrated the complete hTNF gene and T7 promoter in cells expressing the fusion protein and mutations of the T7 promoter site from recovered cells.
authors.affiliation: Institute of Microbial Technology, Sector-39a, Chandigarh-160036, A Constituent Establishment of the Council of Scientific and Industrial Research (CSIR), India.
authors.forename: Gursonika
authors.initials: G
authors.lastname: Binepal
authors.forename: Rajiv Kumar
authors.initials: RK
authors.lastname: Ranjan
authors.forename: Kammara
authors.initials: K
authors.lastname: Rajagopal
mesh_terms: D003470:Culture Media; D004926:Escherichia coli / Q000254:growth & development* / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods; D011994:Recombinant Proteins / Q000235:genetics; D014170:Transformation, Genetic; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics*
chemical_list: D003470:Culture Media; D011994:Recombinant Proteins; C439524:TNF protein, human; D014409:Tumor Necrosis Factor-alpha