Selective Precipitation of Protiens with Ligand-Modified Phospholipids

本提案涉及利用配体修饰磷脂（LMP）亲和沉淀法从复杂生物培养基（微生物细胞培养液、组织匀浆、哺乳动物细胞培养培养液等）中回收蛋白质。配体修饰的磷脂是与配体共价连接的磷脂，配体选择性地与蛋白质分子上的一个位点络合。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于水。溶解后的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。这类配合物通过不同配合物上烃类链之间的疏水相互作用聚集在一起。另外，如果蛋白质上有多个结合位点，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。这些类型的结合导致大的络合物，这些络合物变得不溶并从溶液中沉淀出来。在之前的拨款（CTS-8904192）中，pi证明了这种沉淀确实有效，通过使用dmpe生物素作为配体修饰的磷脂和亲和素（与生物素有四个结合位点）作为蛋白质。为了使LMP溶解，加入了高于CMC浓度的聚氧基表面活性剂C12E8。磷脂溶解到胶束中，这是通过准弹性光散射测量胶束大小的变化推断出来的。当配体修饰的磷脂附着在胶束上时，未观察到沉淀；然而，当溶液被稀释到在亚稳透明溶液中形成亚胶团聚集体时，观察到亲和素的沉淀。一种利用斯摩鲁霍夫斯基动力学计算聚合速率的理论正在发展。由散射引起的浊度测量结果与动力学表达式很好地吻合。新研究建议用该方法检测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），以及单结合位点酶磷酸果糖激酶（PFK）的沉淀。详细介绍了LMPs的合成路线。在新的努力中，将研究杂质蛋白对亲和沉淀的影响，并设计和测试从沉淀聚集体中去除蛋白质的分离方案。

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.