Rattenholl A, Lilie H, Grossmann A, Stern A, Schwarz E, Rudolph R
Institut für Biotechnologie, Martin-Luther-Universität Halle-Wittenberg, Halle/Saale, Germany.
Eur J Biochem. 2001 Jun;268(11):3296-303. doi: 10.1046/j.1432-1327.2001.02232.x.
Nerve growth factor (beta-NGF), a neurotrophin required for the development and survival of specific neuronal populations, is translated as a prepro-protein in vivo. While the presequence mediates translocation into the endoplasmic reticulum, the function of the pro-peptide is so far unknown. As the pro-sequences of several proteins are known to promote folding of the mature part, the renaturation behaviour of recombinant human beta-NGF pro-protein was compared to that of the mature form. Expression of rh-pro-NGF in Escherichia coli led to the formation of inclusion bodies (IBs). The presence of the covalently attached pro-sequence significantly increased the yield and rate of refolding with concomitant disulfide bond formation when compared to the in vitro refolding of mature NGF (rh-NGF). Physicochemical characterization revealed that rh-pro-NGF is a dimer. The pro-peptide could be removed by limited proteolysis with trypsin yielding biologically active, mature rh-NGF. Furthermore, rh-pro-NGF exhibited biological activity in the same concentration range as rh-NGF.
神经生长因子（β-NGF）是特定神经元群体发育和存活所必需的一种神经营养因子，在体内被翻译为前体蛋白原。虽然前导序列介导其转运至内质网，但目前前肽的功能尚不清楚。由于已知几种蛋白质的前序列可促进成熟部分的折叠，因此将重组人β-NGF前体蛋白原的复性行为与成熟形式进行了比较。重组人前体-NGF（rh-pro-NGF）在大肠杆菌中的表达导致包涵体（IBs）的形成。与成熟NGF（rh-NGF）的体外复性相比，共价连接的前序列的存在显著提高了复性产量和速率，并伴随着二硫键的形成。物理化学表征表明，rh-pro-NGF是一种二聚体。用胰蛋白酶进行有限的蛋白水解可去除前肽，产生具有生物活性的成熟rh-NGF。此外，rh-pro-NGF在与rh-NGF相同的浓度范围内表现出生物活性。
