Lamberti Annalisa, Longo Olimpia, Del Vecchio Pompea, Zambrano Nicola, Barone Guido, Russo Tommaso, Arcari Paolo
Dipartimento di Biochimica e Biotecnologie Mediche, Università di Napoli Federico II, Italy.
Biosci Biotechnol Biochem. 2005 Dec;69(12):2395-400. doi: 10.1271/bbb.69.2395.
Rat brain Fe65 and its truncated forms corresponding to the combined PTB1 and PTB2 domains, as well as to the isolated PTB2 domain, were expressed in Escherichia coli and purified from inclusion bodies by affinity chromatography. The recombinant proteins were refolded and judged functionally active by their ability to interact with native APP. Limited proteolysis of recombinant Fe65 and PTB1-2 with trypsin, chymotrypsin and V8 proteases showed that the most sensitive proteoltytic sites were positioned at the level of the interdomain regions comprised between WW/PTB1 and PTB1/PTB2. Secondary structure of the recombinant proteins, evaluated by CD spectroscopy, showed a different degree of unordered structures, the PTB2 domain being the higher organised region. In addition, intrinsic fluorescence measurements of PTB2, indicated that a conformational transition of the protein can be induced by denaturating agents such as GuHCl. These data provide first evidences on the secondary structural levels of Fe65.
大鼠脑Fe65及其对应于组合的PTB1和PTB2结构域以及分离的PTB2结构域的截短形式在大肠杆菌中表达，并通过亲和层析从包涵体中纯化。重组蛋白经复性后，通过与天然APP相互作用的能力判断其功能活性。用胰蛋白酶、胰凝乳蛋白酶和V8蛋白酶对重组Fe65和PTB1-2进行有限的蛋白水解显示，最敏感的蛋白水解位点位于WW/PTB1和PTB1/PTB2之间的结构域间区域水平。通过圆二色光谱法评估的重组蛋白的二级结构显示出不同程度的无序结构，PTB2结构域是组织程度更高的区域。此外，PTB2的内在荧光测量表明，变性剂如盐酸胍可诱导该蛋白发生构象转变。这些数据为Fe65的二级结构水平提供了初步证据。
