Affinity Precipitation of Proteins. Characterization of Some Underlying Mechanisms

Sammanfattning: Two polymers, alginate and Eudragit S-100 were used as reversibly soluble-insoluble ligand carriers for the selective isolation of enzymes and lectins respectively. These two polymers have the inherent property of being precipitated under well defined conditions. Alginate was made insoluble by the addition of Ca2+ ions, and Eudragit S-100 by a pH shift. The polymers were modified with affinity ligands before they could be used for specific recognition of target proteins in affinity precipitation processes. The final result of an affinity precipitation procedure is determined by several underlying mechanisms of which a few were investigated. The initial complex formation between the polymer-ligands and the proteins of interest was studied using dynamic laser light scattering (DLLS). The ligand to target protein ratio was found to be a cruical parameter when the lectin Concanavalin A (Con A) was reacted with p-aminophenyl-a-D-glucopyranoside modified Eudragit S-100. An optimal ratio of 40 ligands (corresponding to 0.5 polymer molecules) per lectin resulted in a maximal initial rate of complex formation. The complex build-up between endo-polygalacturonase and alginate was also determined in a similar way during progress of precipitation. Rheological measurements were used to study the behavior of Eudragit S-100 in solution as well as in a precipitated state. The relatively strong intermolecular network dominated by hydrophobic interactions was broken down as the polymer was modified with affinity ligands. The same tendency was observed for unmodified Eudragit S-100 in the presence of NaCl or protein. Elution from a resolubilized polymer was found to be superior as compared to the recovery of target protein directly from a precipitate. DLLS was used to study the kinetics of the elution of Con A from p-aminophenyl-a-D-glucopyranoside modified Eudragit S-100, initiated by the addition of low molecular weight sugars. The dissociation process followed a simple first order reaction in all cases. The efficiency of an affinity precipitation process for soybean hemagglutinin (SBH) based on Eudragit S-100 was compared with an affinity chromatographic procedure. The yield of SBH from an affinity column was three times higher than the yield from affinity precipitation. Precipitation of SBH was done from protein extracts pretreated to different degrees. A more precessed starting material resulted in a purer product at the expense of yield.