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Soluble expression of disulfide-bonded C-type lectin like domain of human CD93 in the cytoplasm of Escherichia coli

Abstract : CD93 belongs to the group XIV C-type lectin like domain (CTLD) and is closely related to thrombomodulin (CD141). Although CD93 is known to be involved in the regulation of cell adhesion and phagocytosis, its role in innate immunity remains to be fully investigated. Critically, published data about CD141 suggest that CD93 CTLD could be involved in the control of inflammation. In order to address further functional and structural analyses, we expressed human CD93 CTLD with several disulfide bonds in an E. coli expression system. As the E. coli cytoplasm is a reducing compartment, production of disulfide-bond proteins remains a challenge. Hence, we decided to over express CD93 CTLD in commercially available strains of E. coli and co-expressed a sulfhydryl oxidase (Erv1p) and a disulfide isomerase (DsbC). This strategy led to high yield expression of a native form of CD93 CTLD. NMR studies revealed that Ca2 + was not able to bind to CD93 CTLD. We also showed that the recombinant protein could alter LPS pro-inflammatory activity on THP1. This work provides new tool for further functional and structural studies to decipher the functions associated to the CTLD of CD93. This approach may also be used for others members of the group XIV C-type lectin like domain (CD141, CD248 and CLec14A).
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https://hal.univ-reunion.fr/hal-01452891
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Submitted on : Thursday, February 2, 2017 - 12:38:10 PM
Last modification on : Tuesday, September 1, 2020 - 3:07:38 AM

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Brice Nativel, Audrey Figuester, Jessica Andries, Cynthia Planesse, Joël Couprie, et al.. Soluble expression of disulfide-bonded C-type lectin like domain of human CD93 in the cytoplasm of Escherichia coli. Journal of Immunological Methods, Elsevier, 2016, 439, pp.67--73. ⟨10.1016/j.jim.2016.10.003⟩. ⟨hal-01452891⟩

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