Functional Siglec lectin domains from soluble expression in the cytoplasm of Escherichia coli

Pröpster, Johannes M. and Yang, Fan and Ernst, Beat and Allain, Frédéric H.-T. and Schubert, Mario. (2015) Functional Siglec lectin domains from soluble expression in the cytoplasm of Escherichia coli. Protein Expression and Purification, 109. pp. 14-22.

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Official URL: http://edoc.unibas.ch/40918/

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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.
Faculties and Departments:05 Faculty of Science > Departement Pharmazeutische Wissenschaften > Ehemalige Einheiten Pharmazie > Molekulare Pharmazie (Ernst)
UniBasel Contributors:Ernst, Beat and Yang, Fan
Item Type:Article, refereed
Article Subtype:Research Article
Note:Publication type according to Uni Basel Research Database: Journal article
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Last Modified:28 Nov 2017 11:11
Deposited On:11 Aug 2016 07:28

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