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Updated: Jun 12, 2026
Recombinant Protein Expression for Structural Biology in HEK 293F Suspension Cells: A Novel and Accessible Approach
Published on: October 16, 2014
Eukaryotic expression of functionally active recombinant soluble CD83 from HEK 293T cells
Christine Staab1, Petra Mühl-Zürbes, Alexander Steinkasserer
1Department of Dermatology, University Hospital Erlangen, Erlangen, Germany. christine.staab@uk-erlangen.de
Insights
Researchers developed a new method for producing soluble CD83 (sCD83) in human cells, yielding a pure, functional, and glycosylated protein. This eukaryotic expression system overcomes limitations of bacterial production for sCD83, improving its potential therapeutic applications.
Area of Science:
- Immunology
- Molecular Biology
- Biochemistry
Background:
- CD83 is a cell surface protein crucial for mature dendritic cells (DCs).
- Soluble CD83 (sCD83) has shown potential in modulating immune responses, including inhibiting T cell proliferation and experimental autoimmune encephalomyelitis (EAE).
- Bacterial expression of sCD83 leads to lipopolysaccharide (LPS) contamination and lack of functional glycosylation, hindering its therapeutic use.
Purpose of the Study:
- To develop an improved eukaryotic expression system for soluble CD83 (sCD83).
- To obtain LPS-free, functionally glycosylated sCD83.
- To evaluate the functionality of eukaryotic-expressed sCD83 in immune assays.
Main Methods:
- Engineered human embryonic kidney (HEK) 293 T cells for sCD83 expression.
- Purified recombinant sCD83 using eukaryotic expression protocols.
- Assessed sCD83 purity (LPS-free) and glycosylation status.
- Utilized the mixed lymphocyte reaction (MLR) assay to test sCD83 functionality.
Main Results:
- Successfully produced LPS-free, glycosylated sCD83 in HEK 293 T cells.
- Confirmed that all three asparagine residues were at least partially involved in glycosylation.
- Demonstrated reduced DC-mediated T cell proliferation in the MLR assay, consistent with previous findings.
- Established an efficient eukaryotic expression and purification protocol for sCD83.
Conclusions:
- Eukaryotic expression in HEK 293 T cells offers significant advantages over prokaryotic systems for producing functional sCD83.
- The new protocol yields pure, glycosylated sCD83, potentially enhancing its therapeutic efficacy.
- This method provides a more robust platform for studying and utilizing sCD83 in immunological research and therapeutic development.
Abstract:
The cell surface protein CD83 belongs to the immunoglobulin super family and is highly expressed on mature dendritic cells (DCs). A membrane bound and a soluble form of CD83 (sCD83) have been described. Previously, the isolation of a purified recombinant sCD83 molecule from bacterial cultures using high pressure liquid chromatography was reported. This recombinant protein reduced DC-mediated T cell proliferation in vitro and displayed an inhibitory effect in the experimental autoimmune encephalomyelitis (EAE) model. When purifying sCD83 from bacteria, however, a lipopolysaccharide fraction is frequently co-isolated with the recombinant sCD83 protein. Moreover, the subsequent separation of sCD83 from contaminating LPS is usually accompanied by a considerable loss of soluble CD83. A further disadvantage of soluble CD83 expression in prokaryotic cells is the lack of functional glycosylation. To overcome these problems, we developed an alternative strategy to express sCD83 in eukaryotic human embryonic kidney (HEK) 293 T cells. Using this system, we showed that recombinant sCD83 was LPS-free and effectively glycosylated with all three asparagine residues at least partially involved. The functionality of the expressed sCD83 protein was examined using the mixed lymphocyte reaction (MLR) assay, demonstrating a reduced DC-mediated T cell proliferation as previously reported for the sCD83 protein purified from E. coli. Thus, a new protocol for efficient eukaryotic expression and purification of sCD83 was established, which might have several advantages compared to prokaryotic expression systems.
