Mobile phase effects on membrane protein elution during immobilized artificial membrane chromatography

The eluotropic strength of different mobile phases for eluting membrane proteins from immobilized artificial membrane (IAM) chromatography surfaces was studied. Two protein mixtures containing bovine pancreatic PLA(2) were used in this study. Protein mixture I was PLA(2) obtained from Sigma which contained similar to 5-10 major protein bands in electrophoretic gels. Protein mixture II was obtained from fresh bovine pancreatic tissue and contained >100 proteins including the target protein, PLA(2). After adsorbing Sigma PLA(2) to IAM columns, the elution conditions common to conventional chromatographic methods were evaluated for their ability to selectively purify PLA(2). Elution conditions tested were (i) detergent gradients, (ii) salt gradients used during ion-exchange chromatography, (iii) salt conditions used during hydrophobic interaction chromatography, (iv) acetonitrile gradients used during reversed-phase chromatography, and (v) a two-step gradient consisting of first a detergent gradient followed by an acetonitrile gradient. Based on silver-stained electrophoretic protein gels, PLA(2) from protein mixture I was purified to electrophoretic homogeneity with 417-fold increase in specific activity in one step using elution condition (v), and PLA(2) from protein mixture II was purified in one step (660-fold increase in specific activity) using elution condition (iv). Total protein recovery from IAM columns is 70-100%.