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Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction
Published on: May 28, 2012
Endotoxin removing method based on lipopolysaccharide binding protein and polyhydroxyalkanoate binding protein PhaP
Jian Li1, Guanguan Shang, Mingliang You
1Multidisciplinary Research Center, Shantou University, Shantou 515063, Guangdong, China.
New rhLBP-PhaP particles efficiently remove over 90% of endotoxins from water and protein solutions. This novel system offers a non-toxic, easy-to-prepare solution for endotoxin removal in protein purification.
Area of Science:
- Biotechnology
- Biochemistry
- Materials Science
Background:
- Polyhydroxyalkanoates (PHAs) granule associated protein (PhaP) binds hydrophobic polymers.
- Human lipopolysaccharide binding protein (hLBP) is a natural endotoxin receptor.
Purpose of the Study:
- To develop and evaluate a novel fusion protein (rhLBP-PhaP) for endotoxin removal.
- To assess the efficiency of rhLBP-PhaP-coated polyhydroxybutyrate (PHB) particles in endotoxin removal and protein recovery.
Main Methods:
- Genes for hLBP fused with PhaP were expressed in Pichia pastoris.
- Purified rhLBP-PhaP was immobilized on PHB particles.
- Endotoxin removal and protein recovery efficiencies were tested in various solutions and conditions (pH, ionic strength).
Main Results:
- rhLBP-PhaP particles achieved over 90% endotoxin removal efficiency.
- Endotoxin removal and protein recovery were minimally affected by ionic strength.
- pH significantly impacted both endotoxin removal and protein recovery efficiencies.
Conclusions:
- rhLBP-PhaP-coated PHB particles demonstrate high efficiency and ease of preparation for endotoxin removal.
- The system is non-toxic and suitable for endotoxin removal in protein purification processes.
- Further optimization based on pH is recommended for maximal efficiency.
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