Endotoxin Removal Method Based On Lipopolysaccharide Binding Protein And Polyhydroxyalkanoate Binding Protein PhaP

Endotoxins, also called lipopolysaccharides (LPS), are a major component of the outer membrane of Gram-negative bacteria. They are released during bacteria growth, division, or death. Endotoxin is very stable in some kinds of conditions and often forms different molecular aggregation in aqueous soulution. So it is very difficult to remove endotoxin in biological products. Polyhydroxyalkanoates (PHA) is a family of thermo polyesters produced by many microorganisms grown on renewable resources such as sugars or fatty acids. PHA particle associated protein (PhaP) has a strong interaction with PHA or other hydrophobic polymers. Human lipopolysaccharide binding protein (hLBP) is a kind of endotoxin receptor in human plasma. In this research, the gene of fusion protein (rhLBP-PhaP) was expressed in Pichia pastoris GS115 and secreted into cultivate medium. The fusion protein was anchored onto PHB particle surface, which is a member of polyhydroxyalkanoates (PHA). The hLBP-PhaP coated PHB particles were added to water or protein solutions containing endotoxin to study their endotoxin removal and protein recovery efficiencies. The acidic proteins including bovine serum albumin (BSA) and ovalbumin, and basic protein R-chymotrypsinogen were used as model proteins to study the influences of pH and ionic strengths on protein recovery and endotoxin removal. The results demonstrated that endotoxin removal and protein recovery efficiencies were drastically affected by pH changes, but were only slightly affected by ionic strengths. The hLBP-PhaP granules could remove endotoxin with an efficiency of over 90%. All Our results showed that hLBP-PhaP particles have their ease of preparation, high efficiency and nontoxicity. It will be a suitable system for endotoxin removal in the biological products purification industry.