title: Evidence for post-transcriptional regulation of the synthesis of the Escherichia coli HlyB haemolysin translocator and production of polyclonal anti-HlyB antibody.
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 domain, was reduced at least 1000-fold. These results indicate that mRNA structures corresponding to the membrane domain of HlyB greatly limit the post-transcriptional expression of HlyB. When such structures are deleted, or disrupted when part of a larger mRNA, HlyB or the HlyB ATPase domain can be overproduced in milligram quantities and this has facilitated the production of high titre antibodies to HlyB.
authors.affiliation: Institut de Génétique et Microbiologie, Université de Paris XI, Orsay, France.
authors.forename: M A
authors.initials: MA
authors.lastname: Blight
authors.forename: B
authors.initials: B
authors.lastname: Menichi
authors.forename: I B
authors.initials: IB
authors.lastname: Holland
mesh_terms: D000906:Antibodies; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D017123:Bacteriophage T7; D001483:Base Sequence; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D006460:Hemolysin Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D015341:Nucleic Acid Probes; D010446:Peptide Fragments / Q000096:biosynthesis / Q000276:immunology; D010957:Plasmids; D012323:RNA Processing, Post-Transcriptional; D012329:RNA, Bacterial / Q000032:analysis; D012333:RNA, Messenger / Q000096:biosynthesis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid
chemical_list: D000906:Antibodies; D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D006460:Hemolysin Proteins; C065231:HlyD protein, E coli; C050837:Hlyb protein, Bacteria; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D015341:Nucleic Acid Probes; D010446:Peptide Fragments; D012329:RNA, Bacterial; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins