title: Expression, purification, and characterization of a soluble form of human cytomegalovirus glycoprotein B.
abstract: The human cytomegalovirus glycoprotein B gene (gB; gpUL55) was truncated at amino acid 692 and recombined into Autographa californica nuclear polyhedrosis virus (baculovirus). Infection of insect cells with the recombinant baculovirus resulted in high-level expression and secretion of the truncated gB protein (gB-S) into the culture medium. Purification of gB-S by monoclonal antibody affinity chromatography yielded a protein of ca. 200 kDa. Characterization of the 200-kDa purification product indicated that the recombinant gB protein retained many structural and functional features of the viral gB. Comparison of electrophoretic migration patterns in reduced versus nonreduced protein samples and immune blotting analysis with antibodies specific for the amino or carboxy-terminus of gB demonstrated that the recombinant protein was composed of disulfide linked 69 kDa amino terminal and 35-kDa carboxy-terminal fragments. In addition, recognition of the 200-kDa gB-S by a conformational-dependent, oligomer-specific monoclonal antibody suggested that gB-S was properly folded and dimeric. Like the viral gB, gB-S had heparin binding ability. One heparin binding site was found to reside within the 35-kDa carboxy-terminal fragment (aa 492-692). Heparin binding was abolished when gB-S was denatured. These data suggest that gB contains a novel heparin binding motif that is at least partially conformational dependent.
authors.affiliation: Department of Medical Microbiology and Immunology, University of Wisconsin at Madison Medical School 53706-1532, USA.
authors.forename: C
authors.initials: C
authors.lastname: Carlson
authors.forename: W J
authors.initials: WJ
authors.lastname: Britt
authors.forename: T
authors.initials: T
authors.lastname: Compton
mesh_terms: D003587:Cytomegalovirus / Q000378:metabolism*; D006801:Humans; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014759:Viral Envelope Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D014759:Viral Envelope Proteins; C040495:glycoprotein B, Simplexvirus