High-level expression of biologically active, soluble forms of ICAM-1 in a novel mammalian-cell expression system.
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.
Warren
T G
TG
Department of Cellular and Molecular Biochemistry, Monsanto Corporate Research, Monsanto Company, St. Louis, Missouri 63198.
Hippenmeyer
P J
PJ
Meyer
D M
DM
Reitz
B A
BA
Rowold
E
E
Carron
C P
CP
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D006224:Cricetinae; D017077:Culture Media, Conditioned; D018076:DNA, Complementary / Q000235:genetics; D005822:Genetic Vectors; D018799:Intercellular Adhesion Molecule-1 / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016169:Lymphocyte Function-Associated Antigen-1 / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011485:Protein Binding; D011863:Radioimmunoassay; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility
D017077:Culture Media, Conditioned; D018076:DNA, Complementary; D016169:Lymphocyte Function-Associated Antigen-1; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D018799:Intercellular Adhesion Molecule-1