Pröpster Johannes M, Yang Fan, Ernst Beat, Allain Frédéric H-T, Schubert Mario
Institute of Molecular Biology and Biophysics, ETH Zürich, CH-8093 Zürich, Switzerland.
Institute of Molecular Pharmacy, Pharmacenter, University of Basel, CH-4056 Basel, Switzerland; Department of Molecular & Medical Pharmacology, David Geffen School of Medicine at UCLA, CA 90095, USA(1).
Protein Expr Purif. 2015 May;109:14-22. doi: 10.1016/j.pep.2015.01.005. Epub 2015 Jan 24.
Siglecs (sialic acid-binding immunoglobulin-like lectins) are a family of mammalian cell-surface receptors that are involved in cell-cell interactions and signaling functions, primarily expressed on cells of the immune system. Key to their function is their specific binding of distinct sialylated glycan ligands mediated via an N-terminal carbohydrate recognition (lectin) domain. Studies concerning the molecular basis of their individual carbohydrate specificities are rare due to the absence of suitable recombinant expression methods for producing these disulfide-containing proteins in sufficient quantities required for their in-depth in vitro characterization. We established an efficient E. coli-based expression and purification method for Siglec lectin domains, utilizing the trxB gor suppressor strain Rosetta-gami B (DE3) in which proper folding with intact disulfide bonds was achieved in the cytoplasm. The approach is demonstrated for human Siglec-7, -8 and -9 lectin domains and works equally well for expression in nutrient-rich (LB) or minimal growth medium, allowing stable-isotope labeling for NMR studies. The recombinant proteins were properly folded as proven by 2D (1)H-(15)N HSQC NMR spectroscopy and by thermal unfolding followed by CD spectroscopy, and functionally active as confirmed by monitoring ligand binding using NMR titration experiments. Our method enables efficient production of homogeneous and active protein samples in milligram quantities. Its implementation will significantly enhance future structure-function studies of this important class of immune-modulating receptors and will support a variety of applications including screening for natural and synthetic ligands or the development of fluorescently-labeled molecular tools for glycan ligand detection or flow-cytometric cell sorting.
唾液酸结合免疫球蛋白样凝集素（Siglecs）是一类哺乳动物细胞表面受体，参与细胞间相互作用和信号传导功能，主要在免疫系统细胞上表达。其功能的关键在于通过N端碳水化合物识别（凝集素）结构域特异性结合不同的唾液酸化聚糖配体。由于缺乏合适的重组表达方法来大量生产这些含二硫键的蛋白质以进行深入的体外表征，关于它们各自碳水化合物特异性分子基础的研究很少。我们建立了一种基于大肠杆菌的Siglec凝集素结构域高效表达和纯化方法，利用trxB gor抑制菌株Rosetta-gami B（DE3），在细胞质中实现了具有完整二硫键的正确折叠。该方法已在人Siglec-7、-8和-9凝集素结构域上得到验证，在营养丰富的（LB）或基本生长培养基中表达效果相同，可用于NMR研究的稳定同位素标记。通过二维（1）H-（15）N HSQC NMR光谱以及热变性后再进行CD光谱证明重组蛋白正确折叠，通过NMR滴定实验监测配体结合证实其具有功能活性。我们的方法能够高效生产毫克级的均匀且有活性的蛋白质样品。其应用将显著加强对这类重要免疫调节受体未来的结构-功能研究，并支持多种应用，包括筛选天然和合成配体，或开发用于聚糖配体检测或流式细胞术细胞分选的荧光标记分子工具。
