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Linderoth, NA ; Simon, MN ; Rodionova, NA ; Cadene, M ; Laws, WR ; Chait, BT ; Sastry, S

Biophysical analysis of the endoplasmic reticulum-resident chaperone/heat shock protein gp96/GRP94 and its complex with peptide antigen

Biochemistry 40 (5) 1483-1495

par Administrateur - publié le

Abstract :

Animals Vaccinated with heat shock protein (HSP)-peptide complexes develop specific protective immunity against cancers from which the HSPs were originally isolated. This autologous specific immunity has been demonstrated using a number of HSP-peptide antigen complexes. A prototypical HSP-based cancer vaccine is the gp96-peptide antigen complex, which is currently undergoing human clinical trials. Here, we analyzed the structure of a recombinant wild-type and a mutant gp96 protein and their peptide complexes using a number of biophysical techniques, Gel filtration chromatography, dynamic light scattering, and equilibrium analytical ultracentrifugation demonstrated that both a wild-type gp96 and a gp96 mutant lacking a dimerization domain formed higher order structures. More detailed analysis using scanning transmission electron microscopy indicated that both the wild-type and dimerization deletion mutant gp96 protein were organized, unexpectedly, into large aggregates, Size distributions ranged from dimers to octamers and higher.