Pidgeon C, Cai S J, Bernal C
Department of Medicinal Chemistry, School of Pharmacy, Purdue University, West Layfayette, IN 47907, USA.
J Chromatogr A. 1996 Jan 19;721(2):213-30. doi: 10.1016/0021-9673(95)00796-2.
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 PLA2 were used in this study. Protein mixture I was PLA2 obtained from Sigma which contained approximately 5-10 major protein bands in electrophoretic gels. Protein mixture II was obtained from flesh bovine pancreatic tissue and contained > 100 proteins including the target protein, PLA2. After adsorbing Sigma PLA2 to IAM columns, the elution conditions common to conventional chromatographic methods were evaluated for their ability to selectively purify PLA2. 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. PLA2 from protein mixture I was purified to electrophoretic homogeneity with 417-fold increase in specific activity in one step using elution condition (v), and PLA2 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%.
研究了不同流动相从固定化人工膜（IAM）色谱表面洗脱膜蛋白的洗脱能力。本研究使用了两种含有牛胰磷脂酶A2（PLA2）的蛋白质混合物。蛋白质混合物I是从Sigma公司获得的PLA2，在电泳凝胶中含有大约5 - 10条主要蛋白带。蛋白质混合物II是从新鲜牛胰组织中获得的，包含100多种蛋白质，其中包括目标蛋白PLA2。将Sigma PLA2吸附到IAM柱上后，评估了传统色谱方法常用的洗脱条件对选择性纯化PLA2的能力。测试的洗脱条件包括：（i）去污剂梯度；（ii）离子交换色谱中使用的盐梯度；（iii）疏水相互作用色谱中使用的盐条件；（iv）反相色谱中使用的乙腈梯度；（v）由第一步去污剂梯度和第二步乙腈梯度组成的两步梯度。基于银染的蛋白质电泳凝胶，使用洗脱条件（v）一步将蛋白质混合物I中的PLA2纯化至电泳纯，比活性提高了417倍；使用洗脱条件（iv）一步将蛋白质混合物II中的PLA2纯化（比活性提高660倍）。从IAM柱上的总蛋白回收率为70 - 100%。
