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首页> 外文期刊>FEBS Open Bio >Production and characterization of neurosecretory protein GM using Escherichia coli and Chinese Hamster Ovary cells
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Production and characterization of neurosecretory protein GM using Escherichia coli and Chinese Hamster Ovary cells

机译：利用大肠杆菌和中国仓鼠卵巢细胞生产和分泌神经分泌蛋白

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Neurosecretory protein GL (NPGL) and neurosecretory protein GM (NPGM) are paralogs recently discovered in birds and in mammals. The post-translational products of NPGL and of NPGM genes include a signal peptide sequence, a glycine amidation signal, and a dibasic amino acid cleavage site. This suggests that the mature forms of NPGL and of NPGM are small proteins secreted in the hypothalamus and containing an amidated C-terminus. However, endogenous NPGL and NPGM have not yet been identified. Chicken NPGL and NPGM have two highly conserved Cys residues that are likely to form a disulfide bond, while mammalian NPGM has one additional Cys residue located between the two conserved Cys residues and the correct disulfide bond pattern is unclear. In this study, we prepared rat NPGM to elucidate the structure of its mature form. We first expressed the predicted mature NPGM, containing an extra C-terminal Gly, in Escherichia coli SHuffle cells, which are engineered to promote the formation of native disulfide bridges in recombinant proteins. We observed the presence of a disulfide bond between the N-terminal Cys residue and the second Cys residue, while the C-terminal Cys residue was free. Secondly, we transfected a construct containing the entire NPGM open reading frame into Chinese Hamster Ovary cells, and observed that NPGM was cleaved immediately after the signal peptide and that it was secreted into the medium. Furthermore, the protein presented a disulfide bond at the same location observed in recombinant NPGM.
机译：神经分泌蛋白GL（NPGL）和神经分泌蛋白GM（NPGM）是最近在鸟类和哺乳动物中发现的旁系同源物。 NPGL和NPGM基因的翻译后产物包括信号肽序列，甘氨酸酰胺化信号和二价氨基酸切割位点。这表明NPGL和NPGM的成熟形式是下丘脑分泌的小蛋白质，含有酰胺化的C末端。但是，尚未鉴定出内源性NPGL和NPGM。鸡NPGL和NPGM有两个高度保守的Cys残基，很可能形成二硫键，而哺乳动物NPGM有一个额外的Cys残基位于两个保守的Cys残基之间，正确的二硫键模式尚不清楚。在这项研究中，我们准备了大鼠NPGM以阐明其成熟形式的结构。我们首先在大肠杆菌SHuffle细胞中表达了预测的成熟NPGM，其中包含一个额外的C末端Gly，该基因经过工程改造以促进重组蛋白中天然二硫键的形成。我们观察到在N末端Cys残基和第二个Cys残基之间存在二硫键，而C末端Cys残基是游离的。其次，我们将包含整个NPGM开放阅读框的构建体转染到中国仓鼠卵巢细胞中，观察到NPGM在信号肽后立即被切割并且被分泌到培养基中。此外，该蛋白在重组NPGM中观察到的相同位置处呈现二硫键。

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