Selective Precipitation of Protiens with Ligand-Modified Phospholipids

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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.