Difference between revisions of "Part:BBa K811000"

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Cytolysin A (ClyA) Cytolysin A (ClyA) pore forming protein causes cell lysis.

Sequence and Features

Sequence and Features

Usage and Biology

ClyA is a protein native to E. coli, Shigella flexneri, and Salmonella typhi that is capable of forming 13-mer pore complexes in a redox-independent manner. Expression of clyA in the absence of other hemolytic toxins is sufficient to induce hemolysis experimentally, and is therefore considered to be a potent cytolytic agent. Unlike a similar protein, HlyA, ClyA is not synthesized as a protoxin, which requires further posttranslational modifications to become active. ClyA functional immediately following translation of mRNA to protein.

ClyA is a 34kDa protein that is composed primarily of α-helical bundles that form a rod-shaped molecule. The membrane insertion domain is known as a β tongue and is critical for hemolytic activity. If the β tongue is mutated, the hemolytic activity of clyA is abrogated.

The regulation of clyA secretion is not well characterized. To date, the method by which newly synthesized clyA is localized to the periplasm is unknown. All that is known about this process is that the secretion of clyA is not dependent on any known signal sequence, nor does it require cytolytic activity of clyA.

Characterization

The clyA gene was synthesized commercially and cloned into pDawn, a newly developed expression system that induces protein expression in response to irradiation from blue light. The pDawn-clyA construct was then transformed into E. coli. BL21 for subsequent expression.

~50 cfu of BL21 containing pDawn-clyA were plated onto Colombia Agar+ 5% Sheep's Blood plates and incubated for 48 hours under blue light at 25C. As shown below in figure 1, clyA was secreted from BL21 and lysed the red blood cells in the blood agar.

Figure 1: The cytolytic capacity of clyA-his in BL21 cells under blue light induction on 5% Sheep's Blood Agar for 48 hours at 25C

In addition, cytotoxicity was quantified using a Promega Cytotox Fluor Kit. This kit qualifies cell lysis at a fluorescence output of 520nm. It was shown that ClyA successfully lysed cells more effectively at higher concentrations. Furthermore, in both HEK293T and SKBR3 cells, there was a significant cell-dependent effect on cytotoxicity (positive correlation). Comparing 10ug/ml ClyA with the negative control in the cytotoxicity assay against cancerous SKBR3 cells shows over a 14 fold increase in lysis with the presence of ClyA