El Mourabit Haquima, Müller Stefan, Tunggal Lucy, Paulsson Mats, Aumailley Monique
Center for Biochemistry, Faculty of Medicine, University of Cologne, Joseph-Stelzmann-Str. 52, Cologne 50931, Germany.
Protein Expr Purif. 2003 Nov;32(1):95-103. doi: 10.1016/S1046-5928(03)00211-0.
FHL2 (Four and a Half LIM domain-containing protein 2) is a member of a small family of proteins with four LIM domains and an N-terminal half LIM domain. It is an intracellular protein thought to function as an adaptor in the formation of multi-protein complexes involved in signaling. To obtain human FHL2 in amounts allowing further characterization, we evaluated different expression systems and chose to express FHL2 with a His6 tag in insect cells using the baculovirus system. The recombinant protein was highly expressed and could be purified to >98% homogeneity as judged by SDS-PAGE analysis. Purified recombinant FHL2 was used to generate antibodies allowing detection and immunoprecipitation of FHL2 from human cells. Both recombinant and natural FHL2 were characterized by SDS-PAGE and MALDI-TOF mass spectrometry. The molecular mass of the recombinant His6-tagged protein obtained by mass spectrometry was 36,995Da, in good agreement with the apparent mass of 36kDa in SDS-PAGE and slightly higher than the 35,981Da calculated from the sequence of the construct. The measured molecular mass of natural human FHL2 was 32,742Da and the calculated mass was 32,192Da. However, the apparent molecular mass in SDS-PAGE is 41kDa, indicating that the natural protein has an abnormal electrophoretic mobility. The results show that both the recombinant and the natural proteins are post-translationally modified and indicate that such modifications may lead to an abnormal electrophoretic behavior of natural human FHL2.
FHL2（含四个半LIM结构域蛋白2）是一个小蛋白家族的成员，该家族蛋白具有四个LIM结构域和一个N端半LIM结构域。它是一种细胞内蛋白，被认为在参与信号传导的多蛋白复合物形成过程中作为衔接蛋白发挥作用。为了获得足够量的人FHL2以进行进一步的特性分析，我们评估了不同的表达系统，并选择使用杆状病毒系统在昆虫细胞中表达带有His6标签的FHL2。通过SDS-PAGE分析判断，重组蛋白高度表达，并且可以纯化至>98%的纯度。纯化的重组FHL2用于制备抗体，以便从人细胞中检测和免疫沉淀FHL2。重组FHL2和天然FHL2都通过SDS-PAGE和MALDI-TOF质谱进行了表征。通过质谱获得的重组His6标签蛋白的分子量为36,995Da，与SDS-PAGE中36kDa的表观分子量吻合良好，略高于根据构建体序列计算的35,981Da。天然人FHL2的实测分子量为32,742Da，计算分子量为32,192Da。然而，其在SDS-PAGE中的表观分子量为41kDa，表明天然蛋白具有异常的电泳迁移率。结果表明，重组蛋白和天然蛋白都经过了翻译后修饰，并且表明这种修饰可能导致天然人FHL2出现异常的电泳行为。
