Evidence for post-transcriptional regulation of the synthesis of the Escherichia coli HlyB haemolysin translocator and production of polyclonal anti-HlyB antibody

Molecular & General Genetics : MGG
M A BlightI B Holland

Abstract

Extensive attempts were made to overexpress the Escherichia coli haemolysin translocator protein HlyB, and HlyB fragments, utilising high copy number plasmids or hlyB expressed from strong promoters including lambda PR, ptrp and the T7 promoter. Analysis of both cytoplasmic and membrane fractions failed to detect any overexpression of the protein, although all the constructs showed biological activity and there was no evidence of HlyB-induced toxicity. In some constructs, the effect of removing a stem-loop structure, immediately upstream of the start codon and implicated in rho-independent termination of transcription, was tested but this did not lead to over-expression. Nevertheless, analysis of hlyB specific mRNA synthesis revealed that some constructs showed at least a 50-fold increase in mRNA levels, indicating that expression of HlyB may be limited at the translational level. When HlyB was expressed as a hybrid, downstream of LacZ, extremely high level overproduction was then detected in total cell extracts. When the expression of HlyB or HlyB fragments expressed from a T7 promoter was examined, the C-terminal ATPase domain was dramatically overexpressed but the production of fragments encompassing the N-terminal membrane ...Continue Reading

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Citations

Oct 17, 2013·Biochimica Et Biophysica Acta·Sabrina ThomasLutz Schmitt
Jan 23, 2015·Cellular Logistics·Terry L Sasser, Rutilio A Fratti
Jul 14, 2018·Microbiology Spectrum·Sezen MeydanAlexander S Mankin

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