Cloning, expression and characterization of aerolysin from Aeromonas hydrophila in Escherichia coli.
Aerolysin is a toxin (protein in nature) secreted by the strains of Aeromonas spp. and plays an important role in the virulence of Aeromonas strains. It has also found several applications such as for detection of glycosylphosphatidylinositol (GPI)-anchored proteins etc. A. hydrophila is a ubiquitous Gram-negative bacterium which causes frequent harm to the aquaculture. To obtain a significant amount of recombinant aerolysin in the active form, in this study, we expressed the aerolysin in E. coli under the control of T7 RNase promoter. The coding region (AerA-W) of the aerA gene of A. hydrophila XS91-4-1, excluding partial coding region of the signal peptide was cloned into the vector pET32a and then transformed into E. coli b121. After optimizing the expression conditions, the recombinant protein AerA-W was expressed in a soluble form and purified using His. Bind resin affinity chromatography. Recombinant aerolysin showed hemolytic activity in the agar diffusive hemolysis test. Western blot analysis demonstrated good antigenicity of the recombinant protein.
Zhu
Daling
D
The Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China.
Li
Aihua
A
Wang
Jianguo
J
Li
Ming
M
Cai
Taozhen
T
D016980:Aeromonas hydrophila / Q000378:metabolism*; D001427:Bacterial Toxins / Q000276:immunology* / Q000302:isolation & purification; D002846:Chromatography, Affinity; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D052899:Pore Forming Cytotoxic Proteins / Q000276:immunology* / Q000302:isolation & purification; D021382:Protein Sorting Signals / Q000235:genetics / Q000502:physiology; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism
D001427:Bacterial Toxins; D052899:Pore Forming Cytotoxic Proteins; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; C032676:aerolysin