Abstract
mRNA stability is one among many parameters that can potentially affect the level of recombinant gene expression in bacteria. Blocking of the entire prokaryotic transcription machinery by addition of rifampicin is commonly used in protocols for analysis of mRNA stability. Here we show that such treatment can be effectively replaced by a simple, non-invasive method based on removal of the relevant transcriptional inducers and that the mRNA decay can then be followed by qRT-PCR. To establish the methodology we first used the m-toluate-inducible XylS/Pm expression cassette as a model system and analyzed several examples of DNA modifications causing gene expression stimulation in Escherichia coli. The new method allowed us to clearly discriminate whether an improvement in mRNA stability contributes to observed increases in transcript amounts for each individual case. To support the experimental data a simple mathematical fitting model was developed to calculate relative decay rates. We extended the relevance of the method by demonstrating its application also for an IPTG-inducible expression cassette (LacI/Ptac) and by analyzing features of the bacteriophage T7-based expression system. The results suggest that the methodology is useful in elucidating factors controlling mRNA stability as well as other specific features of inducible expression systems. Moreover, as expression systems based on diffusible inducers are almost universally available, the concept can be most likely used to measure mRNA decay for any gene in any cell type that is heavily used in molecular biology research.
Highlights
The use of inducible promoter systems for heterologous protein expression in Escherichia coli is one of the most convenient and actively used methods, accounting for one third of protein production altogether [1,2,3]
We further confirmed the robustness of the method at different levels, as follows: 1) by High-performance Liquid Chromatography (HPLC) it was shown that the inducer is removed from both the media and cells after the filtration; 2) two independent primer pairs targeting the 59 or the 39-terminal end, respectively, were found to give corresponding results during quantitative real time polymerase chain reaction (qRT-PCR) quantifications of the same recombinant mRNA; 3) it was demonstrated that the amounts of an mRNA produced constitutively were not affected by the wash-out procedure itself
To confirm that the wash-out method could be used to analyze mRNA stability we first reinvestigated an earlier study showing strong stimulation of transcript accumulation resulting from specific mutations (LV-2 variant) in the DNA region corresponding to the 59-untranslated region (UTR) mRNA sequence of the bla reporter gene, encoding b-lactamase [14]
Summary
The use of inducible promoter systems for heterologous protein expression in Escherichia coli is one of the most convenient and actively used methods, accounting for one third of protein production altogether [1,2,3]. Together with its positive regulator XylS that is activated by the binding of downstream products in the pathway (alkylbenzoates), the Pm promoter system has been established in our laboratory as a robust model for heterologous expression in various bacterial hosts [5,6,7]. Strong stimulation of the transcript levels has been achieved by mutating the Pm promoter associated 59 untranslated region (UTR) or by fusing translocation signal sequence to a heterologous gene [14,15]
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