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

DIR@IMTECH: CSIR-Institute of Microbial Technology

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Title Expression of synthetic human tumor necrosis factor is toxic to Escherichia coli.
 
Creator Binepal, Gursonika
Ranjan, Rajiv Kumar
Rajagopal, Kammara
 
Subject QH426 Genetics
 
Description 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.
 
Publisher Elsevier Science
 
Date 2012-02-01
 
Type Article
PeerReviewed
 
Format application/pdf
 
Identifier http://crdd.osdd.net/open/1126/1/rajagopal2012.pdf
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
 
Relation http://www.sciencedirect.com/science/article/pii/S0378111911006950
http://crdd.osdd.net/open/1126/