Tang Xia, Chen Jia, Wang Ying, Wang Xianchun
Key Laboratory of Protein Chemistry and Developmental Biology of Ministry of Education, College of Life Sciences, Hunan Normal University, Changsha, 410081 Hunan China.
Springerplus. 2016 Oct 3;5(1):1705. doi: 10.1186/s40064-016-3330-y. eCollection 2016.
Rab3A is a GTP-binding protein and plays critical roles in the regulation of synaptic vesicle exocytosis. Up to date, how Rab3A participates in such a regulatory process is not completely clear.
In this report the Rab3A gene from was cloned and heterologously expressed in using pCold-TF expression vector with folding capacity. Due to the presence of His-tag sequence on the N-terminal side, Rab3A fusion protein was purified to greater than 95 % purity with a single Ni-affinity purification step. After the Rab3A fusion protein was used to immunize mice, an anti-serum against Rab3A with a titer of about 6000 was generated. Western blot analysis indicated that the prepared polyclonal antibody could recognize both Rab3A fusion protein and native Rab3A protein. To remove the tag sequence, thrombin was used to cleave the Rab3A fusion protein, followed by SDS-PAGE to separate the cleavage products. Using the gel protein recovery strategy with a Micro Protein PAGE Recovery Kit, the de-tagged Rab3A protein of electrophoretic purity was prepared.
The present work not only prepared the ground for the study on Rab3A-mediated protein interactions, but also provided systematic experimental methods referable for the similar studies.
Rab3A是一种GTP结合蛋白，在突触小泡胞吐作用的调节中起关键作用。迄今为止，Rab3A如何参与这样一个调节过程尚不完全清楚。
在本报告中，使用具有折叠能力的pCold-TF表达载体克隆了来自[具体来源未给出]的Rab3A基因，并在[具体宿主未给出]中进行了异源表达。由于N端存在His-tag序列，通过单一步骤的镍亲和纯化，Rab3A融合蛋白的纯度达到了95%以上。用Rab3A融合蛋白免疫小鼠后，产生了效价约为6000的抗Rab3A抗血清。蛋白质印迹分析表明，所制备的多克隆抗体能够识别Rab3A融合蛋白和天然Rab3A蛋白。为了去除标签序列，使用凝血酶切割Rab3A融合蛋白，然后通过SDS-PAGE分离切割产物。使用Micro Protein PAGE Recovery Kit的凝胶蛋白回收策略，制备了电泳纯的去标签Rab3A蛋白。
目前的工作不仅为Rab3A介导的蛋白质相互作用研究奠定了基础，也为类似研究提供了可供参考的系统实验方法。
