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Published on: October 16, 2014
High-level expression of biologically active, soluble forms of ICAM-1 in a novel mammalian-cell expression system
T G Warren1, P J Hippenmeyer, D M Meyer
1Department of Cellular and Molecular Biochemistry, Monsanto Corporate Research, Monsanto Company, St. Louis, Missouri 63198.
Insights
Researchers developed a novel mammalian cell expression system to study the LFA-1/ICAM-1 interaction. This system efficiently produces biologically active soluble ICAM-1 proteins for detailed analysis of cellular adhesion.
Area of Science:
- Immunology
- Molecular Biology
- Cell Biology
Background:
- The interaction between Leukocyte Function-associated Antigen-1 (LFA-1) and Intercellular Adhesion Molecule-1 (ICAM-1) is crucial for immune responses.
- Understanding the specific contact sites mediating this interaction is vital for T-cell activation and leukocyte migration.
Purpose of the Study:
- To develop a high-level expression system for producing soluble ICAM-1 proteins.
- To facilitate the study of LFA-1/ICAM-1 binding interactions.
Main Methods:
- Utilized a herpes simplex virus (HSV) VP16 transactivator and IE175 promoter system in BHK cells.
- Engineered expression of foreign genes as fusion proteins with a decapeptide tail for purification.
- Generated cell lines for high-level production of soluble ICAM-1 extracellular domains (sICAM453 and sICAM185).
Main Results:
- Successfully established a mammalian cell expression system for recombinant protein production.
- Produced high levels of biologically active soluble ICAM-1 proteins (sICAM453 and sICAM185).
- Enabled single-step purification of these proteins via antibody affinity chromatography.
Conclusions:
- The developed expression system is effective for generating substantial quantities of soluble ICAM-1.
- The biologically active sICAM-1 proteins are suitable for further investigation of LFA-1 binding.
- This system facilitates research into the molecular mechanisms of cell adhesion and immune cell trafficking.
Abstract:
LFA-1/ICAM-1 interaction is important in facilitating a number of cellular events including antigen-specific T-cell activation and leukocyte transendothelial migration. We are interested in defining residues and contact sites that mediate ICAM-1 interaction with the integrin receptor, LFA-1. To provide sufficient material to facilitate study of the interaction of this ligand-receptor pair, we have developed a new high-level mammalian-cell expression system based on the use of the herpes simplex virus (HSV) VP16 transactivator and the HSV IE175 promoter to direct expression of foreign genes in BHK cells. In this system, the gene of interest is expressed as a fusion protein with a carboxyl terminal decapeptide tail to aid in identification, quantitation, and affinity purification of recombinant protein. This system allowed rapid generation of cell lines producing high levels of levels of soluble proteins corresponding to the full-length extracellular (sICAM453) and the amino terminal two immunoglobulin domains (sICAM185) of ICAM-1. Both sICAM453 and sICAM185 were biologically active and were purified in a single step from conditioned media by antibody affinity chromatography.
