Properties and purification of an active biotinylated lactose permease from Escherichia coli.

A simplified approach for purification of functional lactose permease from Escherichia coli is described that is based on the construction of chimeras between the permease and a 100-amino acid residue polypeptide containing the biotin acceptor domain from the oxaloacetate decarboxylase of Klebsiella pneumoniae [Cronan, J. E., Jr. (1990) J. Biol. Chem. 265, 10327-10333]. Chimeras were constructed with a factor Xa protease site and the biotin acceptor domain in the middle cytoplasmic loop (loop 6) or at the C terminus of the permease. Each construct catalyzes active lactose transport in cells and right-side-out membrane vesicles. Moreover, the constructs are biotinylated in vivo, and in both chimeras, the factor Xa protease site is accessible from the cytoplasmic surface of the membrane. Both biotinylated permeases bind selectively to immobilized monomeric avidin and are eluted with free biotin in a high state of purity, and the loop 6 chimera catalyzes active transport after reconstitution into proteoliposomes. The methodology described should be applicable to other membrane proteins. IMAGES:

本研究报道了一种从大肠杆菌（Escherichia coli）中纯化功能性乳糖通透酶的简化策略，该策略通过在通透酶与一段源自肺炎克雷伯菌（Klebsiella pneumoniae）草酰乙酸脱羧酶的、含100个氨基酸残基的生物素受体结构域（biotin acceptor domain）多肽之间构建嵌合体实现[Cronan, J. E., Jr. (1990) J. Biol. Chem. 265, 10327-10333]。研究人员分别在通透酶的中间胞质环（环6）以及C端构建了带有Xa因子蛋白酶（factor Xa protease）位点与生物素受体结构域的嵌合体。每种构建体均可在细胞以及外翻膜囊泡（right-side-out membrane vesicles）中催化活性乳糖转运。此外，这些构建体可在体内发生生物素化修饰，且两种嵌合体的Xa因子蛋白酶位点均可从膜的胞质侧被接触到。两种生物素化的通透酶均可选择性结合至固定化单体亲和素（monomeric avidin），并可通过游离生物素洗脱获得高纯度产物；其中环6嵌合体在重构至蛋白脂质体（proteoliposomes）后仍可催化活性乳糖转运。本研究所描述的方法有望推广应用于其他膜蛋白。图像：