Selective Precipitation of Protiens with Ligand-Modified Phospholipids

用配体修饰的磷脂选择性沉淀蛋白质

• 批准号: 9200823
• 负责人: Peter Kilpatrick
• 金额: $ 36.96万
• 依托单位: North Carolina State University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-08-01 至 1997-01-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9200823&HistoricalAwards=false
• 关键词: Selective; Precipitation; Protiens; Ligand; Modified

This proposal is concerned with recovery of proteins from complex biological media (microbial cell broths, tissue homogenates, mammalian cell culture broths, etc.) by affinity precipitation using ligand-modified phospholipids (LMP). Ligand modified phospholipids are phospholipids to which is covalently attached a ligand which selectively complexes to a site on a protein molecule. Ligand modified phospholipids are solubilized in water by hydrophobic association with nonionic surfactants. The solubilized LMP/surfactant complex then binds with the active site on the protein to form a protein- phospholipid-surfactant complex. Such complexes aggregate by virtue of hydrophobic interactions among hydrocarbon chains on different complexes. Also another LMB/surfactant complex can bind to the protein of a LMB/surfactant/protein complex if there is more than one binding site on the protein. Associations of these types lead to large complexes which become insoluble and precipitate out of solution. In a previous grant (CTS-8904192), the PIs demonstrated that this precipitation does work by using DMPE-biotin as the ligand modified phospholipid and avidin (with four binding sites to biotin) as the protein. To solubilized the LMP, the polyethoxy surfactant C12E8 was added in concentrations above the CMC. The phospholipid solubilized itself into the micelles as was inferred by changes in micellar size as measured by quasi-elastic light scattering. When the ligand modified phospholipid was attached to the micelles, precipitation was not observed; however, when the solution was diluted to presumably form sub-micelle aggregates in a metastable, transparent solution, precipitation of the avidin was observed. A theory for the rate of aggregate formation was developing using Smoluchowski kinetics. Measurements of the turbidity due to scattering were shown to be in good agreement with the kinetic expressions. The new research suggests examining the precipitation by this method of two polyclonal (goat IgG) antibodies with two binding sites, anti- biotin antibody (ABA) and anti-digoxin antibody (ADA), and a single binding site enzyme phosphofructokinase (PFK). The synthesis routes for the LMPs are detailed. In the new effort, the effects of impurity proteins on the affinity precipitation will be examined, and isolation schemes to remove the protein from the precipitating aggregate will be devised and tested.

该建议与使用配体改性的磷脂（LMP）亲和力沉淀相关的复杂生物学培养基（微生物细胞匀浆，哺乳动物细胞培养汤等）的蛋白质涉及蛋白质的回收。 配体修饰的磷脂是磷脂，共价附着在蛋白质分子上的位点上有选择性地将其复合到位点。 配体修饰的磷脂通过疏水与非离子表面活性剂的关联在水中溶解。 然后，溶解的LMP/表面活性剂复合物与蛋白质上的活性位点结合，形成蛋白质磷脂表面表面活性剂复合物。 这种复合物是由于不同复合物上烃链之间的疏水相互作用而聚集的。 如果蛋白质上有多个结合位点，则另一种LMB/表面活性剂复合物可以与LMB/表面活性剂/蛋白质复合物的蛋白质结合。 这些类型的关联导致大型复合物，这些复合物变得不溶于溶液并沉淀出来。 在先前的赠款（CTS-8904192）中，PIS证明了这种降水确实通过使用DMPE-Biotin作为配体修饰的磷脂和Avidin（与生物素的四个结合位点）作为蛋白质起作用。 为了溶解LMP，在CMC上方添加了多乙基表面活性剂C12E8。 正如准弹性光散射测得的胶束大小的变化所推断的那样，磷脂溶解在胶束中。 当配体修饰的磷脂附着在胶束上时，未观察到降水。但是，当将溶液稀释至可能在亚稳态，透明溶液中形成亚微米骨料时，观察到抗生物素的沉淀。 骨料形成速率的理论是使用Smoluchowski动力学发展的。 由于散射引起的浊度的测量与动力学表达非常吻合。 新研究表明，通过这种方法检查了两种结合位点的多克隆（山羊IgG）抗体的降水，抗生物素抗体（ABA）和抗二醇抗体（ADA）以及一种单个结合位点酶酶酶酶（PFK）。 详细介绍了LMP的合成路线。 在新的工作中，将检查杂质蛋白对亲和沉淀的影响，并将设计和测试从沉淀骨料中去除蛋白质的分离方案。