This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Obukowicz, M G Articles by Krivi, G G Search for Related Content PubMed PubMed Citation Articles by Obukowicz, M G Articles by Krivi, G G Agricola Articles by Obukowicz, M G Articles by Krivi, G G

 Previous Article  |  Next Article 

Appl Environ Microbiol. 1992 May; 58(5): 1511-1523

Enhanced heterologous gene expression in novel rpoH mutants of Escherichia coli.

Monsanto Corporate Research, Monsanto Company, St. Louis, Missouri 63198.

ABSTRACT

Extragenic temperature-resistant suppressor mutants of an rpoD800 derivative of Escherichia coli W3110 were selected at 43.5 degrees C. Two of the mutants were shown to have a phenotype of enhanced accumulation of heterologous proteins. Genetic mapping of the two mutants showed that the mutation conferring temperature resistance resided in the rpoH gene. P1-mediated transduction of the rpoD+ gene into both of the rpoD800 rpoH double mutants resulted in viable rpoH mutants, MON102 and MON105, that retained temperature resistance at 46 degrees C, the maximum growth temperature of W3110. The complete rpoH gene, including the regulatory region, from MON102, MON105, and the parental W3110 was cloned and sequenced. Sequencing results showed that a single C----T transition at nucleotide 802 was present in both MON102 and MON105, resulting in an Arg(CGC)----Cys(TGC) substitution at amino acid residue 268 (R-268-C; this gene was designated rpoH358). Heterologous protein accumulation levels in both MON102 and MON105, as well as in rpoH358 mutants constructed in previously unmanipulated W3110 and JM101, were assessed and compared with parental W3110 and JM101 levels. Expression studies utilizing the recA or araBAD promoter and the phage T7 gene 10L ribosome-binding site (g10L) showed that increased accumulation levels of a number of representative heterologous proteins (i.e., human or bovine insulin-like growth factor-1, bovine insulin-like growth factor-2, prohormone of human atrial natriuretic factor, bovine placental lactogen, and/or bovine prolactin) were obtained in the rpoH358 mutants compared with the levels in the parental W3110 and JM101. The mechanism of enhanced heterologous protein accumulation in MON102 and MON105 was unique compared with those of previously described rpoH mutants. Pulse-chase and Northern (RNA) blot analyses showed that the enhanced accumulation of heterologous proteins was not due to decreased proteolysis but was instead due to increased levels of the respective heterologous mRNAs accompanied by increased synthesis of the respective heterologous proteins. The plasmid copy number remained unaltered.

This article has been cited by other articles:

• Noel, S., Herman, A., Johnson, G. A., Gray, C. A., Stewart, M. D., Bazer, F. W., Gertler, A., Spencer, T. E. (2003). Ovine Placental Lactogen Specifically Binds to Endometrial Glands of the Ovine Uterus. Biol. Reprod. 68: 772-780 [Abstract] [Full Text]  
• Herman, A., Helman, D., Livnah, O., Gertler, A. (1999). Ruminant Placental Lactogens Act as Antagonists to Homologous Growth Hormone Receptors and as Agonists to Human or Rabbit Growth Hormone Receptors. J. Biol. Chem. 274: 7631-7639 [Abstract] [Full Text]  
• Helman, D., Staten, N. R., Grosclaude, J., Daniel, N., Nespoulous, C., Djiane, J., Gertler, A. (1998). Novel Recombinant Analogues of Bovine Placental Lactogen. G133K AND G133R PROVIDE A TOOL TO UNDERSTAND THE DIFFERENCE BETWEEN THE ACTION OF PROLACTIN AND GROWTH HORMONE RECEPTORS. J. Biol. Chem. 273: 16067-16074 [Abstract] [Full Text]  
• Helman, D., Staten, N. R., Byatt, J., Grosclaude, J., McKinnie, R. E., Djiane, J., Gertler, A. (1997). Site-Directed Mutagenesis of Recombinant Bovine Placental Lactogen at Lysine-73 Leads to Selective Attenuation of Its Somatogenic Activity. Endocrinology 138: 4069-4080 [Abstract] [Full Text]  
• Vashdi-Elberg, D., Staten, N. R., Sakal, E., McKinnie, R. E., Djiane, J., Krivi, G. G., Gertler, A. (1996). Selective Modification of Recombinant Bovine Placental Lactogen by Site-directed Mutagenesis at Its C Terminus. J. Biol. Chem. 271: 5558-5564 [Abstract] [Full Text]