Papala Agnes, Sylvester Marc, Dyballa-Rukes Nadine, Metzger Sabine, D'Haese Jochen
Institute of Cell Biology, Heinrich Heine University Duesseldorf, Duesseldorf, Germany.
Institute of Biochemistry and Molecular Biology, University of Bonn, Bonn, Germany.
Protein Expr Purif. 2017 Jun;134:25-37. doi: 10.1016/j.pep.2017.03.017. Epub 2017 Mar 18.
CapG is an actin-binding protein, which is overexpressed in a variety of tumors, i.e. breast, ovarian, pancreatic and lung carcinoma. We successfully expressed human CapG in the wild type strain X-33 of the methylotrophic yeast Pichia pastoris (P. pastoris), which does not express endogenous CapG, in order to characterize this protein in more detail. After mechanical cell lysis, debris was centrifuged and the soluble protein was precipitated with ammonium sulfate. This protein pellet was dialyzed and used for CapG purification. Ca-dependent exposure of hydrophobic sites allowed single step and selective elution from a Phenyl Sepharose™ matrix. 3.5 mg CapG/10 g wet biomass were isolated and showed a Ca-sensitive and dose-dependent capping activity of actin in a fluorometric assay. In P. pastoris, CapG is located at actin patches, actin cables and arranges along the budding neck. The proliferation rate and morphology of the yeast cells are not influenced by the interaction of CapG with actin. The modification pattern of human CapG from P. pastoris and human carcinoma cells is highly similar. We validated most of the known post-translational modifications and found three new phosphorylation and nine new acetylation sites by mass spectrometry. The N-terminus is acetylated or truncated. Truncated CapG is not phosphorylated at the residues S10, T212 and S337. First mutagenesis experiments indicate an N-terminal acetylation dependent C-terminal phosphorylation.
CapG是一种肌动蛋白结合蛋白，在多种肿瘤中过表达，如乳腺癌、卵巢癌、胰腺癌和肺癌。我们成功地在不表达内源性CapG的甲基营养型酵母毕赤酵母（P. pastoris）的野生型菌株X-33中表达了人CapG，以便更详细地表征该蛋白。机械细胞裂解后，将碎片离心，并用硫酸铵沉淀可溶性蛋白。将该蛋白沉淀透析后用于CapG的纯化。疏水位点的钙依赖性暴露允许从苯基琼脂糖™基质中进行单步和选择性洗脱。每10克湿生物质分离出3.5毫克CapG，并且在荧光测定中显示出对肌动蛋白的钙敏感且剂量依赖性的封端活性。在毕赤酵母中，CapG位于肌动蛋白斑点、肌动蛋白丝束上，并沿出芽颈部排列。酵母细胞的增殖速率和形态不受CapG与肌动蛋白相互作用的影响。来自毕赤酵母和人癌细胞的人CapG的修饰模式高度相似。我们验证了大多数已知的翻译后修饰，并通过质谱发现了三个新的磷酸化位点和九个新的乙酰化位点。N端被乙酰化或截短。截短的CapG在S10、T212和S337残基处未被磷酸化。首次诱变实验表明N端乙酰化依赖于C端磷酸化。
