Shanmugabalaji Venkatasalam, Douet Véronique, Agne Birgit, Kessler Felix
Laboratory of Plant Physiology, University of Neuchatel.
Institut fur Biochemie und Biotechnologie, Martin-Luther-Universitat Halle-Wittenberg.
J Vis Exp. 2018 Nov 1(141). doi: 10.3791/58532.
Chloroplast biogenesis requires the import of thousands of nucleus-encoded proteins into the plastid. The import of these proteins depends on the translocon at the outer (TOC) and inner (TIC) chloroplast membranes. The TOC and TIC complexes are multimeric and probably contain yet unknown components. One of the main goals in the field is to establish the complete inventory of TOC and TIC components. For the isolation of TOC-TIC complexes and the identification of new components, the preprotein receptor TOC159 has been modified N-terminally by the addition of the tandem affinity purification (TAP) tag resulting in TAP-TOC159. The TAP-tag is designed for two sequential affinity purification steps (hence "tandem affinity"). The TAP-tag used in these studies consists of a N-terminal IgG-binding domain derived from Staphylococcus aureus Protein A (ProtA) followed by a calmodulin-binding peptide (CBP). Between these two affinity tags, a tobacco etch virus (TEV) protease cleavage site has been included. Therefore, TEV protease can be used for gentle elution of TOC159-containing complexes after binding to IgG beads. In the protocol presented here, the second Calmodulin-affinity purification step was omitted. The purification protocol starts with the preparation and solubilization of total cellular membranes. After the detergent-treatment, the solubilized membrane proteins are incubated with IgG beads for the immunoisolation of TAP-TOC159-containing complexes. Upon binding and extensive washing, TAP-TOC159 containing complexes are cleaved and released from the IgG beads using the TEV protease whereby the S. aureus IgG-binding domain is removed. Western blotting of the isolated TOC159-containing complexes can be used to confirm the presence of known or suspected TOC and TIC proteins. More importantly, the TOC159-containing complexes have been used successfully to identify new components of the TOC and TIC complexes by mass spectrometry. The protocol that we present potentially allows the efficient isolation of any membrane-bound protein complex to be used for the identification of yet unknown components by mass spectrometry.
叶绿体生物发生需要将数千种核编码蛋白导入质体。这些蛋白的导入依赖于叶绿体外膜（TOC）和内膜（TIC）上的转运体。TOC和TIC复合物是多聚体，可能还包含未知成分。该领域的主要目标之一是确定TOC和TIC成分的完整清单。为了分离TOC-TIC复合物并鉴定新成分，前体蛋白受体TOC159在N端进行了修饰，添加了串联亲和纯化（TAP）标签，得到TAP-TOC159。TAP标签设计用于两个连续的亲和纯化步骤（因此称为“串联亲和”）。这些研究中使用的TAP标签由源自金黄色葡萄球菌蛋白A（ProtA）的N端IgG结合结构域和钙调蛋白结合肽（CBP）组成。在这两个亲和标签之间，包含一个烟草蚀纹病毒（TEV）蛋白酶切割位点。因此，TEV蛋白酶可用于在与IgG磁珠结合后温和洗脱含TOC159的复合物。在此处介绍的方案中，省略了第二个钙调蛋白亲和纯化步骤。纯化方案从制备和溶解总细胞膜开始。经过去污剂处理后，将溶解的膜蛋白与IgG磁珠孵育，以免疫分离含TAP-TOC159的复合物。结合并充分洗涤后，使用TEV蛋白酶切割含TAP-TOC159的复合物并从IgG磁珠上释放，从而去除金黄色葡萄球菌IgG结合结构域。对分离的含TOC159的复合物进行蛋白质免疫印迹可用于确认已知或疑似TOC和TIC蛋白的存在。更重要的是，含TOC159的复合物已成功用于通过质谱鉴定TOC和TIC复合物的新成分。我们提出的方案有可能高效分离任何膜结合蛋白复合物，用于通过质谱鉴定未知成分。
