Endogenous glutathione-binding proteins of insect cell lines: Characterization and removal from glutathione S-transferase (GST) fusion proteins

After affinity purification on immobilized glutathione, insect-cell-derived glutathione S-transferase (GST) fusion proteins contain variable amounts o f protein contaminants of about 23-24 kDa. We have isolated these glutathio ne-binding proteins from the widely used Sf9 and Hi5 insect cell lines and characterized them by LC-MS and N-terminal sequencing. Based on the observa tion that these proteins have higher affinity for glutathione than GST fusi ons, we have found that by using differential elution conditions the amount of such contaminants in GST fusion preparations can be strongly reduced di rectly during the affinity purification step. The main interest of these re sults is that they are not restricted to a specific construct, but rather t hey seem to apply to various insect-cell-derived GST fusions. (C) 2000 Acad emic Press.