An engineered chaperonin caging a guest protein: Structural insights and potential as a protein expression tool.
The structure of a chaperonin caging a substrate protein is not quite clear. We made engineered group II chaperonins fused with a guest protein and analyzed their structural and functional features. Thermococcus sp. KS-1 chaperonin alpha-subunit (TCP) which forms an eightfold symmetric double-ring structure was used. Expression plasmids were constructed which carried two or four TCP genes ligated head to tail in phase and a target protein gene at the 3' end of the linked TCP genes. Electron microscopy showed that the expressed gene products with the molecular sizes of ~120 kDa (di-TCP) and ~230 kDa (tetra-TCP) formed double-ring complexes similar to those of wild-type TCP. The tetra-TCP retained ATPase activity and its thermostability was significantly higher than that of the wild type. A 260-kDa fusion protein of tetra-TCP and green fluorescent protein (GFP, 27 kDa) was able to form the double-ring complexes with green fluorescence. Image analyses indicated that the GFP moiety of tetra-TCP/GFP fusion protein was accommodated in the central cavity, and tetra-TCP/GFP formed the closed-form similar to that crystallographically resolved in group II chaperonins. Furthermore, it was suggested that caging GFP expanded the cavity around the bottom. Using this tetra-TCP fusion strategy, two virus structural proteins (21-25 kDa) toxic to host cells or two antibody fragments (25-36 kDa) prone to aggregate were well expressed in the soluble fraction of Escherichia coli. These fusion products also assembled to double-ring complexes, suggesting encapsulation of the guest proteins. The antibody fragments liberated by site-specific protease digestion exhibited ligand-binding activities.
Furutani
Masahiro
M
Sekisui Chemical Co. Ltd., Hyakuyama 2-1, Shimamoto-cho, Mishima-gun, Osaka 618-8589, Japan. furutani002@sekisui.jp
Hata
Jun-Ichi
J
Shomura
Yasuhito
Y
Itami
Keisuke
K
Yoshida
Takao
T
Izumoto
Yoshitaka
Y
Togi
Akiko
A
Ideno
Akira
A
Yasunaga
Takuo
T
Miki
Kunio
K
Maruyama
Tadashi
T
D000251:Adenosine Triphosphatases / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry; D001483:Base Sequence; D018833:Chaperonins / Q000737:chemistry*; D002850:Chromatography, Gel; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000378:metabolism; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000737:chemistry; D007091:Image Processing, Computer-Assisted; D047468:Immunoprecipitation; D008024:Ligands; D008274:Magnesium / Q000737:chemistry; D008854:Microscopy, Electron; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D017510:Protein Folding; D040901:Proteomics / Q000295:instrumentation / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013696:Temperature; D019712:Thermococcus / Q000378:metabolism; D013997:Time Factors
D019843:Archaeal Proteins; D008024:Ligands; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D018833:Chaperonins; D008274:Magnesium