In vitro production of an active neurotrophic factor, neuregulin-1 : qualitative comparison of different cell-free translation systems
In vitro production of an active neurotrophic factor, neuregulin-1 : qualitative comparison of different cell-free translation systems
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活性神经营养因子 neregulin-1 的体外生产：不同无细胞翻译系统的定性比较
DOI：
10.1016/j.neulet.2011.04.036
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发表时间：
2011
期刊：
影响因子：
2.5
通讯作者：
Nawa H
中科院分区：
文献类型：
--
作者：
Wang R;Iwakura Y;Araki K;Sotoyama H;Takei N;Nawa H
许多神经营养因子及其变体的个体生物活性，是由选择性剪接和蛋白水解加工产生的，通常仍有待表征。细菌蛋白质生产结合蛋白质折叠和纯化是获得活性神经营养因子的常规方法；然而，这个过程是耗时的，适当的蛋白质在体外折叠有时是不可预测的。在这里，我们研究了三种不同的无细胞翻译系统：网织细胞裂解液，Hela细胞裂解液和小麦胚芽提取物，这可能使我们能够在单管中产生生物活性因子。以i型神经调节蛋白1 β a3为例，我们从其带有Cap核苷酸和/或内部核糖体进入位点（IRES）的mrna中提取神经调节蛋白1，并比较了这些系统翻译产物的产量和生物活性。在Hela无细胞体系中，IRES+ mRNA的蛋白产量最高，而背景翻译在小麦胚芽体系中最低。然而，两种翻译产物的生物活性都是适度的或可以忽略不计的。网状细胞裂解液中产生神经调节蛋白1，产率为19 pmol/mL (~ 500 ng/mL)；此外，它还能磷酸化ErbB4受体，并能与硫酸肝素结合。这些结果表明，网织细胞裂解物翻译系统在体外产生活性神经营养因子，并有助于放射性标记或初步评估新的神经营养因子及其变异。
The individual biological activities of many neurotrophic factors and their variants, which are produced by alternative splicing and proteolytic processing, often remain to be characterized. Bacterial protein production combined with protein refolding and purification is a conventional procedure to obtain active neurotrophic factors; however, the procedure is time consuming and appropriate protein refoldingin vitrois sometimes unpredictable. Here we examined three distinct cell-free translation systems: reticulocyte lysate, Hela cell lysate and wheat germ extract, which may allow us to produce biologically active factors in a single tube. Taking type-I neuregulin-1 beta3 as an example, we produced neuregulin-1 protein from its mRNAs flanked by Cap nucleotide and/or internal ribosome entry site (IRES) and compared the yields and biological activity of translation products from these systems. The protein yield from IRES+ mRNA was highest in the Hela cell-free system, while background translation was lowest in the wheat germ system. The biological activity of both translation products was modest or negligible, however. Neuregulin-1 protein was produced in reticulocyte lysate at yields of 19 pmol/mL (∼500 ng/mL); furthermore, it was potent at phosphorylating ErbB4 receptor and able to bind to heparin sulfate. These results demonstrate that the reticulocyte lysate translation system produces active neurotrophic factorsin vitroand is useful for radiolabeling or preliminary assessment of novel neurotrophic factors and their variants.