Department of Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford, CA, USA.
J Neurosci Methods. 2011 Sep 15;200(2):121-8. doi: 10.1016/j.jneumeth.2011.06.020. Epub 2011 Jul 2.
In this report, we describe a novel method for producing mature and biologically active mono-biotinylated nerve growth factors (mBtNGF) that can be used for single molecule studies of real-time movement of neurotrophins within axons of neurons. We inserted an AviTag sequence into the C-terminal of the full length mouse preproNGF cDNA and cloned the fusion construct into a pcDNA3.1 mammalian expression vector. We also subcloned the Escherichia coli biotin ligase, BirA, into a pcDNA3.1 vector. These two plasmids were then transiently co-expressed in HEK293FT cells. As a result, the AviTag located in the C-terminal of preproNGF was selectively ligated to a single biotin by BirA. The prepro sequence of NGF was subsequently cleaved within the cell. Mature mono-biotinylated NGF (mBtNGF) was secreted into cell culture media and was purified using Ni resin. We carried out activity assays and our results showed that mBtNGF retained biological activities that were comparable to normal NGF purified from mouse sub maxillary glands. We further verified the biotinylation efficiency of mBtNGF and the level of non-biotinylated NGF was virtually undetectable in the final preparation. Finally, by conjugating to quantum-dot streptavidin, mBtNGF was successfully used for single molecule study of axonal NGF trafficking in neurons.
在本报告中，我们描述了一种生产成熟且具有生物活性的单生物素化神经生长因子（mBtNGF）的新方法，该方法可用于实时研究神经营养因子在神经元轴突内的单个分子运动。我们将 AviTag 序列插入全长小鼠前蛋白 NGF cDNA 的 C 末端，并将融合构建体克隆到 pcDNA3.1 哺乳动物表达载体中。我们还将大肠杆菌生物素连接酶 BirA 亚克隆到 pcDNA3.1 载体中。然后，这两个质粒在 HEK293FT 细胞中瞬时共表达。结果，位于前蛋白 NGF C 末端的 AviTag 被 BirA 选择性地连接到单个生物素上。NGF 的前蛋白序列随后在细胞内被切割。成熟的单生物素化 NGF（mBtNGF）被分泌到细胞培养物中，并使用 Ni 树脂进行纯化。我们进行了活性测定，结果表明 mBtNGF 保留了与从小鼠颌下腺中纯化的正常 NGF 相当的生物活性。我们进一步验证了 mBtNGF 的生物素化效率，并且在最终制剂中几乎无法检测到非生物素化的 NGF。最后，通过与量子点链霉亲和素缀合，mBtNGF 成功地用于神经元轴突 NGF 运输的单分子研究。
