title: Functional Siglec lectin domains from soluble expression in the cytoplasm of Escherichia coli.
abstract: 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.
authors.affiliation: Institute of Molecular Biology and Biophysics, ETH Zürich, CH-8093 Zürich, Switzerland.
authors.forename: Johannes M
authors.initials: JM
authors.lastname: Pröpster
authors.affiliation: 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).
authors.forename: Fan
authors.initials: F
authors.lastname: Yang
authors.affiliation: Institute of Molecular Pharmacy, Pharmacenter, University of Basel, CH-4056 Basel, Switzerland.
authors.forename: Beat
authors.initials: B
authors.lastname: Ernst
authors.affiliation: Institute of Molecular Biology and Biophysics, ETH Zürich, CH-8093 Zürich, Switzerland. Electronic address: allain@mol.biol.ethz.ch.
authors.forename: Frédéric H-T
authors.initials: FH
authors.lastname: Allain
authors.affiliation: Institute of Molecular Biology and Biophysics, ETH Zürich, CH-8093 Zürich, Switzerland; Department of Molecular Biology, University of Salzburg, A-5020 Salzburg, Austria(1). Electronic address: mario.schubert@sbg.ac.at.
authors.forename: Mario
authors.initials: M
authors.lastname: Schubert
mesh_terms: D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003593:Cytoplasm / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D066244:Proton Magnetic Resonance Spectroscopy; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D063265:Sialic Acid Binding Immunoglobulin-like Lectins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility
chemical_list: D011994:Recombinant Proteins; D063265:Sialic Acid Binding Immunoglobulin-like Lectins
keywords: Carbohydrate recognition; Disulfide bond; Glycan-binding receptor; I-type lectin; Recombinant expression; Siglec