唾液酸是一种含有九个碳的单糖的衍生物。广泛的存在于动物体的组织内。尤其是人类的大脑,含有高浓度的唾液酸。这些唾液酸被认为是神经节苷脂的传递递质,对神经传导具有重要意义。唾液酸在体内生物膜上的转运是由一种具有多个跨膜螺旋的膜蛋白NanT介导的。NanT蛋白是SLC17家族转运体蛋白的一员,它的功能丧失会导致扎拉疾病以及婴儿唾液酸积累紊乱。然而唾液酸转运体蛋白的结构以及其底物转运机制目前都是未知的。在这篇文章中,我们克隆并表达了NanT蛋白。结合亲和层析以及凝胶阻滞层析等手段,我们获得了高纯度的NanT目的蛋白。我们的结果为进一步研究NanT的结构以及其底物转运机制提供了很好的基础。 Sialic acids are nine-carbon sugar acids, and are found widely distributed in animal tissues. In human, the brain contains high concentration of sialic acids, which are vital for neural transmis-sion. The transport of sialic acids in vivo is carried out by the multi-pass membrane protein, NanT, a membrane of the SLC17 transporter family. The loss of function of NanT will cause salla disease and infantile sialic acid storage disorder. However, the structure and mechanism of the sialic acids transporter remain unknown. In this study, we clone and express the NanT protein. Combining the affinity and size-exclusion chromatography, we obtain the high purity of the target protein. Our results provide a basis for further exploring the structure and mechanism of the sialic acids transporter, NanT.
我们首先采用Ni2+亲和层析柱(NTA)进行蛋白纯化,先用binding buffer ( 20 mM HEPES pH 7.5, 150 mM NaCl,1 mM DDM)平衡柱子,然后把离心上清液缓慢流经柱子。上样完成后,用binding buffer冲洗柱子20个柱体积,然后用washing buffer ( 20 mM HEPES pH 7.5, 150 mM NaCl, 1 mM DDM, 20 mM iminazole)洗脱10个柱体积去除杂蛋白。最后用elution buffer ( 20 mM HEPES pH 7.5, 150 mM NaCl, 1 mM DDM, 300 mM iminazole)洗脱目的蛋白。
2.2.3. NanT蛋白的凝胶阻滞层析
收集的目的蛋白液用浓缩管(milipore)浓缩至1 ml,加入30 μl TEV酶进行柱上过夜酶切来除去组氨酸标签,酶切产物用Superdex™200 (GE Healthcare)分子筛柱子进行进一步纯化,缓冲液为 20 mM HEPES pH 7.5,150 mM NaCl,1 mM DDM,流速为0.5 ml/min,最后用SDS-PAGE鉴定蛋白纯度。
黄尚谕,史青茹. 唾液酸转运体蛋白NanT的分子克隆,异源表达及纯化 Molecular Cloning, Heterologous Expression, and Purification of the Sialic Acid Transporter NanT[J]. 生物医学, 2016, 06(04): 25-30. http://dx.doi.org/10.12677/HJBM.2016.64004
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