Multidisciplinary Research Center, Shantou University, Shantou 515063, Guangdong, China.
Biomacromolecules. 2011 Mar 14;12(3):602-8. doi: 10.1021/bm101230n. Epub 2011 Jan 24.
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.
聚羟基烷酸酯 (PHA) 颗粒相关蛋白 PhaP 对 PHA 和其他疏水聚合物具有很强的亲和力。人脂多糖结合蛋白 (hLBP) 是血浆中天然内毒素受体。在这项研究中，编码 hLBP 与 PhaP 融合的基因在毕赤酵母 GS115 中表达，以生产融合蛋白。纯化的 rhLBP-PhaP 融合蛋白固定在聚羟基丁酸酯 (PHB) 颗粒上，PHB 是微生物聚羟基烷酸酯 (PHA) 的成员。将 rhLBP-PhaP 涂层的 PHB 颗粒添加到含有内毒素的水和蛋白质溶液中，以研究它们对内毒素的去除和蛋白质的回收效率。还使用酸性蛋白质（包括牛血清白蛋白 (BSA)、卵清蛋白和碱性蛋白 α-糜蛋白酶原）作为模型蛋白质，研究了不同蛋白质溶液中离子强度和 pH 值对内毒素去除和蛋白质回收的影响。结果表明，rhLBP-PhaP 颗粒可以去除超过 90%的内毒素。所有内毒素去除和蛋白质回收效率仅受离子强度的轻微影响，但受 pH 值变化的剧烈影响。我们的结果表明，rhLBP-PhaP 颗粒具有高效、易于制备和无毒的特点，将成为蛋白质纯化行业中去除内毒素的合适系统。
