Consler T G, Persson B L, Jung H, Zen K H, Jung K, Privé G G, Verner G E, Kaback H R
Howard Hughes Medical Institute, Department of Physiology, University of California, Los Angeles 90024-1662.
Proc Natl Acad Sci U S A. 1993 Aug 1;90(15):6934-8. doi: 10.1073/pnas.90.15.6934.
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.
本文描述了一种从大肠杆菌中纯化功能性乳糖通透酶的简化方法，该方法基于通透酶与一种含有肺炎克雷伯氏菌草酰乙酸脱羧酶生物素受体结构域的100个氨基酸残基多肽之间的嵌合体构建[克罗南，J.E.，Jr.（1990）《生物化学杂志》265，10327 - 10333]。嵌合体在通透酶的中间细胞质环（环6）或C末端构建了一个因子Xa蛋白酶切割位点和生物素受体结构域。每个构建体在细胞和外翻膜囊泡中催化活性乳糖转运。此外，构建体在体内被生物素化，并且在两种嵌合体中，因子Xa蛋白酶切割位点都可从膜的细胞质表面接近。两种生物素化的通透酶都选择性地结合到固定化的单体抗生物素蛋白上，并以高纯度用游离生物素洗脱，并且环6嵌合体在重组到蛋白脂质体后催化活性转运。所描述的方法应该适用于其他膜蛋白。
