LLO PEST free
Description
Recombinant Listeriolysin O s a single polypeptide protein encoded by the hlyA gene and composed of 529 residues. PEST sequence is 19 amino acids peptide located at the protein NH 2-terminus, that targets the toxin for degradation. This motif is essential for bacterial virulence.
Product Specs
Product Science Overview
Listeriolysin O (LLO) is a cholesterol-dependent cytolysin produced by the pathogenic bacterium Listeria monocytogenes. This protein plays a crucial role in the pathogenicity of L. monocytogenes, enabling the bacterium to escape from the phagosomal compartment of host cells and proliferate within the cytoplasm . The PEST sequence, a proline, glutamic acid, serine, and threonine-rich region, is known to regulate the cytotoxicity of LLO. The PEST-free recombinant version of LLO is engineered to lack this sequence, thereby altering its regulatory properties.
Listeriolysin O is synthesized as a precursor protein composed of 529 amino acid residues. It possesses a typical signal sequence at its N-terminus, which is cleaved to produce the mature protein . The mature LLO is secreted as a monomer and functions by forming pores in the host cell membrane, facilitating the escape of L. monocytogenes from the phagosome into the cytoplasm .
The recombinant LLO, including the PEST-free variant, is typically produced by expressing the hlyA gene (encoding LLO) in Escherichia coli. The purification process involves a one-step method that yields a significant amount of functional LLO. This method ensures the stability and activity of the protein, which can be retained at 4°C for over a year . The recombinant LLO can be tagged with either an N-terminus or C-terminus His tag to facilitate purification .
The synthetic route for producing recombinant LLO involves cloning the hlyA gene into an expression vector suitable for E. coli. The expression of the gene is induced, and the protein is subsequently purified using affinity chromatography. The PEST-free variant is engineered by deleting the PEST sequence from the hlyA gene before cloning .
Listeriolysin O’s pore-forming activity is essential for its function in facilitating the escape of L. monocytogenes from the phagosome. The protein interacts with cholesterol in the host cell membrane, leading to pore formation. This activity is crucial for the bacterium’s intracellular survival and proliferation . The absence of the PEST sequence in the recombinant variant does not significantly affect its pore-forming ability but may alter its regulatory properties .
The recombinant LLO, particularly the PEST-free variant, has several applications in research and biotechnology. It is used as a tool to study the intracellular lifecycle of L. monocytogenes and the host-pathogen interactions. Additionally, it has potential applications in vaccine development and as an adjuvant in immunotherapy .
