Endotoxin removing method based on lipopolysaccharide binding protein and polyhydroxyalkanoate binding protein PhaP.
Polyhydroxyalkanoates (PHAs) granule associated protein PhaP has a strong affinity to PHA and other hydrophobic polymers. Human lipopolysaccharide binding protein (hLBP) is a natural endotoxin receptor in plasma. In this study, genes encoding hLBP fused with PhaP were expressed in Pichia pastoris GS115 for production of the fusion protein. The purified rhLBP-PhaP fusion protein was immobilized on particles of polyhydroxybutyrate (PHB), which is a member of microbial polyhydroxyalkanoates (PHA). The rhLBP-PhaP-coated PHB particles were added to endotoxin containing water and protein solutions to study their endotoxin removal and protein recovery efficiencies. The influences of ionic strengths and pH on endotoxin removal and protein recovery in different protein solutions were also studied using acidic proteins including bovine serum albumin (BSA), ovalbumin, and basic protein α-chymotrypsinogen as model proteins. The results showed that rhLBP-PhaP particles could remove endotoxin with an efficiency of over 90%. All endotoxin removal and protein recovery efficiencies were only slightly affected by ionic strengths but were drastically affected by pH changes. Our results demonstrated that rhLBP-PhaP particles with their high efficiency, ease of preparation, and nontoxicity will be a suitable system for endotoxin removal in the protein purification industry.
Multidisciplinary Research Center, Shantou University, Shantou 515063, Guangdong, China.
Jian
J
Li
Guanguan
G
Shang
Mingliang
M
You
Siwu
S
Peng
Zhihui
Z
Wang
Hongning
H
Wu
Guo-Qiang
GQ
Chen
D000209:Acute-Phase Proteins / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism / Q000627:therapeutic use*; D004340:Drug Contamination / Q000517:prevention & control; D004345:Drug Industry / Q000592:standards; D004731:Endotoxins / Q000302:isolation & purification*; D006801:Humans; D055767:Immobilized Proteins; D008562:Membrane Glycoproteins / Q000378:metabolism; D008722:Methods; D054813:Polyhydroxyalkanoates / Q000378:metabolism; D000091402:Prohibitins; D011506:Proteins / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins; D000099231:Lipopolysaccharide-Binding Protein
D000209:Acute-Phase Proteins; D002352:Carrier Proteins; D004731:Endotoxins; D055767:Immobilized Proteins; D008562:Membrane Glycoproteins; D054813:Polyhydroxyalkanoates; D000091402:Prohibitins; D011506:Proteins; D011993:Recombinant Fusion Proteins; D000099231:Lipopolysaccharide-Binding Protein; C000717328:PHB protein, human