EVIDENCE FOR POST TRANSCRIPTIONAL REGULATION OF THE SYNTHESIS OF THE ESCHERICHIA-COLI HLYB HEMOLYSIN TRANSLOCATOR AND PRODUCTION OF POLYCLONAL ANTI-HLYB ANTIBODY

Extensive attempts were made to overexpress the Escherichia coli haemo lysin translocator protein HlyB, and HlyB fragments, utilising high co py number plasmids or hlyB expressed from strong promoters including l ambda pR ptrp and the T7 promoter. Analysis of both cytoplasmic and me mbrane fractions failed to detect any overexpression of the protein, a lthough 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 codo n and implicated in rho-independent termination of transcription, was tested but this did not lead to overexpression. 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 expr essed as a hybrid, downstream of LacZ, extremely high level overproduc tion 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 produc tion of fragments encompassing the N-terminal membrane domain, was red uced at least 1000-fold. These results indicate that mRNA structures c orresponding to the membrane domain of HlyB greatly limit the post-tra nscriptional expression of HlyB. When such structures are deleted, or disrupted when part of a larger mRNA, HlyB or the HlyB ATPase domain c an be overproduced in milligram quantities and this has facilitated th e production of high titre antibodies to HlyB.