A 6xHistidine tag insertion at the N-terminal signal peptide A42 and G43 may interfere with BrkA secretion and expression in Escherichia coli DH5α
Abstract
BrkA is a type Vα autotransporter on the gram-negative human pathogen Bordetella pertussis. BrkA mediates serum resistance and cellular adherence; however, the secretion mechanism of BrkA has yet to be fully elucidated. We aimed to investigate the secretion of BrkA into the periplasm and extracellular space. We inserted a 6xHis-tag at the N-terminal signal peptide A42 and G43 on brkA-containing pDO6935. We then conducted an immunoblot assay using our test plasmid but did not observe BrkA secretion in Escherichia coli (E. coli) DH5α. Follow-up DNA sequencing of the newly constructed 6xHis-tagged BrkA coding plasmid showed that a frameshift mutation had been introduced in the brkA sequence, which resulted in a truncated and possibly unstable protein product. Since we were unable to isolate clones containing the desired 6xHis tag insertion in BrkA, we speculate that the 6xHis tag insertion near the signal sequence cleavage site may have resulted in the production of a toxic BrkA variant, which may have stalled in the Sec translocon resulting in cell death.
