Expression of synthetic human tumor necrosis factor is toxic to Escherichia coli.

Binepal, Gursonika and Ranjan, Rajiv Kumar and Rajagopal, Kammara (2012) Expression of synthetic human tumor necrosis factor is toxic to Escherichia coli. Gene, 493 (1). pp. 155-60. ISSN 1879-0038

PDF rajagopal2012.pdf - Published Version Restricted to Registered users only Download (717Kb) | Request a copy

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.

Item Type:	Article
Additional Information:	Copyright of this article belongs to Elsevier Science.
Uncontrolled Keywords:	Synthetic human TNF-α; Fusion protein; T7 promoter; Overlap forward-primer-walk polymerase chain reaction.
Subjects:	Q Science > QH Natural history > QH426 Genetics
Depositing User:	Dr. K.P.S.Sengar
Date Deposited:	21 Mar 2012 16:13
Last Modified:	21 Mar 2012 16:13
URI:	http://crdd.osdd.net/open/id/eprint/1126

Actions (login required)

View Item