Abstract
Intercellular adhesion molecule 1 (ICAM-1) is a membrane-bound glycoprotein expressed on endothelial cells and cells of the immune system. Human ICAM-1 mediates adhesion and migration of leucocytes, and is implicated in inflammatory pathologies, autoimmune diseases and in many cancer processes. Additionally, ICAM-1 acts as receptor for pathogens like human rhinovirus and Plasmodium falciparum malaria parasites. A group of related P. falciparum erythrocyte membrane protein 1 (PfEMP1) domains, the DBLβ, mediates ICAM-1 binding of P. falciparum-infected erythrocytes. This ICAM‑1-binding phenotype has been suggested to be involved in the development of cerebral malaria. However, more studies identifying cross-reactive antibody and ICAM-1-binding epitopes and the establishment of a clinical link between DBLβ expression and e.g. cerebral malaria are needed before the DBLβ domains can be put forward as vaccine candidates and go into clinical trials. Such studies require availability of functional recombinant ICAM-1 in large quantities. In this study, we compared recombinant ICAM-1 expressed in HEK293 and COS-7 cells with mouse myeloma NS0 ICAM-1 purchased from a commercial vendor in terms of protein purity, yield, fold, ability to bind DBLβ, and relative cost. We present a HEK293 cell-based, high-yield expression and purification scheme for producing inexpensive, functional ICAM‑1. ICAM-1 expressed in HEK293 is applicable to malaria research and can also be useful in other research fields.
Highlights
Intercellular adhesion molecule 1 (ICAM-1) is a member of the immunoglobulin (Ig) superfamily and is expressed by endothelial cells and leucocytes as a membrane-bound protein containing five extracellular Ig-like domains (D1-D5), a trans-membrane domain, and a cytoplasmic domain
Transfected HEK293 cells were grown for 6 days before harvesting ICAM-1-FcHEK293 from the supernatant
Supernatant ICAM-1-FcHEK293 could be detected by dot blotting before and after buffer exchange and concentration (Figure 1A) but was not visible by SDS-PAGE (Figure 1B, lane 2)
Summary
ICAM-1 is a member of the immunoglobulin (Ig) superfamily and is expressed by endothelial cells and leucocytes as a membrane-bound protein containing five extracellular Ig-like domains (D1-D5), a trans-membrane domain, and a cytoplasmic domain. ICAM‐1 mediates adhesion and migration of leukocytes by binding to leukocyte function-associated antigen-1 (LFA‐1) and macrophage antigen-1 (Mac-1) [1,2] It is implicated in inflammatory pathologies, autoimmune diseases, and many cancer processes [3]. More studies linking ICAM‐1-adhering IEs to severe disease such as cerebral malaria and identifying ICAM-1binding PfEMP1 epitopes (not least epitopes inducing adhesion-inhibitory antibodies) are needed before DBLβ3_D4 can be put forward as a vaccine candidate. Achievement of this goal depends heavily on the availability of large quantities of high-quality recombinant ICAM-1. We present a HEK293 cell-based, high-yield expression and purification scheme for producing inexpensive, functionally intact ICAM-1 able to bind the P. falciparum antigen PFD1235w-DBLβ3_D4
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