Expression and functional characterization of recombinant human vascular cell adhesion molecule-1 (VCAM1) synthesized by baculovirus-infected insect cells.
Vascular cell adhesion molecule-1 (VCAM1) is a cell surface glycoprotein produced by the vascular endothelium, as well as on macrophage-like and dendritic cell types, in response to certain inflammatory stimuli. VCAM1 interacts with the integrin VLA4 present on mononuclear leukocytes. We have isolated the cDNA for VCAM1 using RT-PCR by screening a cDNA library from IL-1 beta-activated human endothelial cells. To obtain large quantities of VCAM1 for structural and functional studies, we have produced this protein in insect cells using a baculovirus expression system. Insect cells infected with recombinant virus synthesized human VCAM1 at levels exceeding 3% of total cellular protein following 72 h postinfection. VCAM1-expressing insect cells were shown to bind specifically to a variety of VLA4 expressing cell lines (Jurkat, THP-1, U937). Thus, recombinant VCAM1 protein produced in the baculovirus expression system was localized to the cell surface and was biologically active. Large-scale availability of this adhesion protein should enhance efforts toward the discovery of new antiadhesive (anti-inflammatory and antiatherogenic) therapeutics.
Stoltenborg
J K
JK
Department of Biotechnology, Du Pont Merck Pharmaceutical Company, Wilmington, Delaware 19880-0400.
Straney
R A
RA
Tritch
R J
RJ
Mackin
W M
WM
George
H J
HJ
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D001681:Biological Assay; D002448:Cell Adhesion; D015815:Cell Adhesion Molecules / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D002478:Cells, Cultured; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D005455:Fluorescent Antibody Technique; D006801:Humans; D008969:Molecular Sequence Data; D009036:Moths / Q000166:cytology; D011994:Recombinant Proteins / Q000096:biosynthesis; D019010:Vascular Cell Adhesion Molecule-1
D015815:Cell Adhesion Molecules; D018076:DNA, Complementary; D011994:Recombinant Proteins; D019010:Vascular Cell Adhesion Molecule-1