Figure 5

Recovery of recombinant mouse CCT complex by co-expression of 8 proteins in the same insect cell. A) Top panel, cell lysates from cells expressing mouse CCT subunits 1-8 (lanes 1-8, respectively). Lane 9, uninfected cells; Lane 10, lysate from cells coexpressing the CCT subunits from the same baculovirus genome; M, marker protein lane. Relative migration of CCT subunits is indicated on the left hand side of the gel. Loci used in the expression were CCT1-polyhedrin, CCT2-p10, CCT3-egt, CCT4-39K, CCT5-gp37, CCT6-odv-e56, CCT7-ctx, CCT8-orf51. Middle panel, Immunoblot using pan-specific antiserum against mouse CCT, sample order as in top panel. Bottom panel, Immunoblot using monospecific antiserum against mouse CCT theta (CCT8), sample order as in top panel. B) Fractions 12 to 22 (of 34) numbered from the top of a 10%-40%(w/w) sucrose gradient. CCT complex proteins peak in the expected ratio in fractions 16 and 17. Relative migrations expected for the CCT proteins are indicated on the left of the gel. Middle panel, immunoblot using pan-specific antiserum against mouse CCT subunits. Lower panel, electron micrographs of negatively stained sample from the 23.5S peak fraction collected from the sucrose gradient. Ring structures consistent with CCT complexes can be detected. Bar = 25 nm.