The cost of purification in biotechnological processes usually amount to 50-90% of the total cost of the product. Therefore there is a considerable incentive to improve the method of purification. After the discovery of metal affinity, its application has been at the fields of chromatography(IMAC), metal affinity aqueous two phase extraction, precipitation methods etc.
In this work, the protein precipitation using the metal ion was studied. The used proteins were phosphoproteins such as ovalbumin (0∼2 phosphoryl groups/molecule) and phosvitin (100∼126 phosphoryl groups/molecule). According to the IMAC and the metal ion affinity aqueous two phase extraction, the phosphoryl group has ferric ion affinity and the histidine has cupper ion affinity. It is essential to know the characteristics of accessible surface of protein and metal to examine the separation mechanism clearly.
According to the experimental result, when the ferric ion for the precipitant was used the phosvitin was precipitated above 95% by metal ion affinity at low pH, but when the cupper ion was used it was precipitated above 98% by not metal ion affinity but other ion interaction between the surface accessible side chains of peptides in protein and metal ion above isoelectric point. Ovalbumin was precipitated above 90% by ferric ion above isoelectric point like phosvitin-cupper ion reaction. In the case of phosvitin-cupper ion and ovalbumin-ferric ion reaction the major factors to affect the precipitation were the acidic groups(aspartic acid, glutamic acid, histidine) attached the surface accessible side chains of peptides in protein.
We may obtain the various pure proteins at one time by the very simple process using the natures of the metal-protein affinity and(or) the acidic side chain of peptide in the protein.
