title: Reconstitution of the human chaperonin CCT by co-expression of the eight distinct subunits in mammalian cells.
abstract: The eukaryotic cytosolic chaperonin CCT (chaperonin-containing TCP-1) assists folding of newly synthesized polypeptides. The fully functional CCT is built from two identical rings, each composed of single copies of eight distinct subunits. To study the structure and function of the CCT complex and the role of each subunit, a rapid and efficient method for preparing a recombinant CCT complex is needed. In this work, we established an efficient expression and purification method to obtain human recombinant CCT. BHK-21 cells were infected with a vaccinia virus expressing T7 RNA polymerase and transfected with eight plasmids, each encoding any one of the eight CCT subunits in the T7 RNA polymerase promoter/terminator unit. The CCT1 subunit was engineered to carry a hexa-histidine tag or FLAG tag in the internal loop region. Three days later, cells were harvested for purification of the CCT complex through tag-dependent affinity chromatography and gel filtration. The purified recombinant CCT complexes were indistinguishable from the endogenous CCT purified from HeLa cells in terms of morphology and function. In conclusion, the co-expression system established in this study should be a simple and powerful tool for reconstitution of a large multi-subunit complex.
authors: Department of Materials Science and Chemistry, Graduate School of Engineering, University of Hyogo, Himeji 671-2280, Japan.
authors: Kodai
authors: K
authors: Machida
authors: Mamiko
authors: M
authors: Masutani
authors: Tominari
authors: T
authors: Kobayashi
authors: Satoshi
authors: S
authors: Mikami
authors: Yuri
authors: Y
authors: Nishino
authors: Atsuo
authors: A
authors: Miyazawa
authors: Hiroaki
authors: H
authors: Imataka
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D056404:Chaperonin Containing TCP-1 / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D017510:Protein Folding; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D014616:Vaccinia virus / Q000235:genetics
chemical_list: D021122:Protein Subunits; D011994:Recombinant Proteins; D056404:Chaperonin Containing TCP-1