Spontaneous cAMP-dependent derepression of gene expression in stationary phase plays a role in recombinant expression instability

Gene
T H GrossmanM S Osburne

Abstract

E. coli recombinant expression systems that utilize lac operon control elements to modulate gene expression are known to produce some amount of uninduced (leaky) gene expression. Previously, we showed that high levels of uninduced gene expression was a major cause of instability in the pET expression system. We show here that the pET system, in which the phage T7 RNA polymerase gene is expressed via lac operon control elements, exhibits leaky expression that increases markedly as cells grown in complex medium enter stationary phase. Moreover, we found that this phenomenon occurs with the chromosomal lac operon as well. Further investigation revealed that stationary phase leaky expression requires cyclic AMP, and that substantial leaky expression could be effected in log phase cells by adding cyclic AMP and acetate at pH6.0. Finally, a comparison of otherwise isogenic cya and wild-type hosts showed that expression stability and plasmid maintenance in the cya host is greatly enhanced, even when cells are passaged repeatedly in non-selection medium. These findings both provide a method to enhance the stability of lac-based recombinant expression systems, and suggest that derepression of the lac operon in the absence of inducer may...Continue Reading

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Citations

Sep 19, 2012·Applied Microbiology and Biotechnology·Christian DetzelJoachim Jose
Aug 6, 2008·Applied Biochemistry and Biotechnology·Dasari Venkata Krishna RaoAdibhatla Kali Satya Bhujanga Rao
Jan 12, 2011·Molecular Biotechnology·Charles GauciMarshall W Lightowlers
Jan 1, 2014·Analytical Biochemistry·Sarvenaz SarabipourKalina Hristova
Jan 13, 2004·Biochemical and Biophysical Research Communications·Jingxing OuAnlong Xu
Mar 28, 2003·Biochemical and Biophysical Research Communications·Yujuan ZhengJiazuan Ni
Jun 5, 1999·Journal of Immunological Methods·M S OsburneT H Grossman
Jan 5, 2002·Journal of Biotechnology·Anant PatkarFriedrich Srienc
Oct 6, 1999·Current Opinion in Biotechnology·F Baneyx
Oct 3, 2002·Protein Expression and Purification·Maria L B MagalhãesDiógenes S Santos
Jan 3, 2003·Protein Expression and Purification·Rafael G SilvaDiógenes S Santos
Oct 9, 2003·Kidney International·Serge M GislerHeini Murer
Apr 4, 2006·Acta Crystallographica. Section F, Structural Biology and Crystallization Communications·Ana Luiza VivanDiógenes Santiago Santos
May 10, 2005·Immunology and Cell Biology·María José Dus Santos, Andrés Wigdorovitz
Feb 14, 2006·BMC Biotechnology·Yiannis IoannouAnisur Rahman
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Sep 12, 2015·Amino Acids·Wooseok KoHyun Soo Lee
Aug 12, 2009·New Biotechnology·Rahul NairSriram Padmanabhan
Apr 25, 2009·International Journal of Biological Macromolecules·Raquel Cristina SchwankeLuiz Augusto Basso

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