Soluble Expression Of Recombinant Proteine Mediated By Chaperonin GroEL/GroES And The Folding-assembiy Mechanism

Chaperonin GroEL is one of the in-depth studied molecular chaperones. It functionally forms a central cavity (named Anfinsen cage) assisted by the co-chaperone GroES and ATP to provide a compartment in which a single protein molecule is allowed to fold unimpaired by aggregation. However, the phenomenon that the obligate GroEL-dependent substrates are highly enriched with complex topological structures still needs to be elucidated. Recombinant human interferon-gamma (rhIFN-γ) is an important pharmaceutical protein but with special structure. For de novo protein synthesis in E. coli, overexpression of rhIFN-γ was found to accumulate as cytoplasmic inclusion bodies driven by the hydrophobic forces that derived from the peculiar structure. This thesis aims to seek whether the readily self-associated rhIFN-y can achieve soluble expression assisted by GroEL, and preliminarily explore the GroEL-mediated folding-assembly pathway.Firstly, the rhIFN-γ gene was fused to a pET-28a expression vector, and the resulting vector was named pET-IFN-γ. The vectors of pET-IFN-γ and pGro7encoding chaperonin GroEL/GroES were cotransformed into E. coli BL21to construct the GroEL/GroES and rhIFN-y coexpression system.The fermentation conditions (e.g. culture temperature, inducer concentration) were investigated, and the soluble rhIFN-y expression was successfully achieved at indispensable low temperature and high GroEL/GroES level. Interestingly, a significantly positive correlation was found between the ratio of supernatant to precipitate of rhIFN-y and the amount of chaperonin, indicating GroEL/GroES could effectively enhance soluble rhIFN-y expression. The optimized conditions were as follows:the overnight cultured seed liquid was transformed into150ml2×YT medium with1%inoculum, and meanwhile,0.5g/L of L-arabinose was added to induce the GroEL/GroES expression;0.2mM of IPTG was added to induce the rhIFN-y expression when OD6oo reached0.6; After further growth at25℃and180 rpm for8h, the cells were harvested. As a result, the soluble expression of rhIFN-y was improved accordingly by2.2-fold than the control.The purified soluble rhIFN-y with Ni-affinity chromatography was shown to effectively fold into the correct conformation that was identical to the native rhIFN-y via the conformational characterization such as secondary structure (CD spectrum), tertiary structure (fluorescence spectrum), and quaternary structure (SEC and DSS cross-linking). Taking the enzyme-linked immunosorbent assay (ELISA) for immunoreactivity, coexpressing GroEL/GroES obtained72.91mg/L of active rhIFN-y, which fortunately opened up a new opportunity for the production of active rhIFN-y in pharmaceutical field.After achieving active expression of rhIFN-y mediated by GroEL/GroES, a series of methods were applied to understand its folding-assembly process. Protein-protein interactions between rhIFN-y and chaperonin were analyzed using the yeast two-hybrid system, demonstrating chaperonin GroEL/GroES strongly interacted with rhIFN-γ to increase its soluble expression. Coexpressing other molecular chaperones (e.g. trigger factor, DnaK system) showed that rhIFN-y folded to native state only upon transfering to GroEL/GroES, which meant rhIFN-γ was an obligate GroEL-dependent (Class Ⅲ) substrate. Combining the specific structure and folding pathway of rhIFN-y, the "conformational maintenance" of GroEL contributing to the assembly process of oligomeric proteins was proposed, and molecular dynamic simulations (e.g. ZDOCK, RDOCK and CHARMm) were used to lend preliminary support to the "conformational maintenance" with hydrogen bonds, ionic bonds and hydrophobic interactions.