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.

该提案涉及使用配体修饰磷脂 (LMP) 通过亲和沉淀从复杂生物介质（微生物细胞肉汤、组织匀浆、哺乳动物细胞培养肉汤等）中回收蛋白质。 配体修饰的磷脂是共价连接有配体的磷脂，该配体选择性地复合至蛋白质分子上的位点。 配体修饰的磷脂通过与非离子表面活性剂的疏水缔合而溶解在水中。 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-表面活性剂复合物。 这种复合物凭借不同复合物上的烃链之间的疏水相互作用而聚集。 如果蛋白质上存在多个结合位点，另一种LMB/表面活性剂复合物也可以与LMB/表面活性剂/蛋白质复合物的蛋白质结合。 这些类型的缔合导致形成大的复合物，该复合物变得不溶并从溶液中沉淀出来。 在之前的资助 (CTS-8904192) 中，PI 证明，通过使用 DMPE-生物素作为配体修饰的磷脂和亲和素（具有四个生物素结合位点）作为蛋白质，这种沉淀确实起作用。 为了溶解 LMP，添加浓度高于 CMC 的聚乙氧基表面活性剂 C12E8。 通过准弹性光散射测量的胶束尺寸的变化推断磷脂自身溶解到胶束中。 当配体修饰的磷脂附着到胶束上时，没有观察到沉淀；然而，当溶液被稀释以可能在亚稳定透明溶液中形成亚胶束聚集体时，观察到抗生物素蛋白沉淀。 使用 Smoluchowski 动力学开发了一种关于聚集体形成速率的理论。 由散射引起的浊度测量结果与动力学表达式非常一致。 新研究建议通过这种方法检查两种具有两个结合位点的多克隆（山羊 IgG）抗体、抗生物素抗体（ABA）和抗地高辛抗体（ADA）以及单结合位点磷酸果糖激酶（PFK）的沉淀。 详细介绍了 LMP 的合成路线。 在新的努力中，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聚集体中去除蛋白质的分离方案。