Efficient production of endotoxin depleted bioactive α-hemolysin of uropathogenicEscherichia coli

Uropathogenic E. coli (UPEC), especially associated with severe urinary tract infections (UTI) pathologies, harbors an important virulence factor known as α-hemolysin (110 kDa). Hemolytic activity of α-hemolysin (HlyA) requires modification (acylation) of two lysine residues of HlyA by HlyC, part of operon hlyCABD. Most of the previous studies had used whole operon hlyCABD and gene tolC cloning for the production of active α-hemolysin. Studies involving α-hemolysin are limited due to the cumbersome and manual method of purification for this toxin. Here, we report a simple method for production of both active and inactive recombinant α-hemolysin by cloning only hlyA and hlyC genes of operon hlyCABD. Presence of both active and inactive α-hemolysin would be advantageous for functional characterization. After translation, the yield of the purified α-hemolysin was 1 mg/200 ml. Functionality of the recombinant α-hemolysin protein was confirmed using hemolytic assay. This is the first report of the production of active and inactive recombinant α-hemolysin for functional studies.