Structure And Function Research Of Regulatory Factor From Cellulosome In Clostridium Thermocellum

Clostridium thermocellum is one of microbes with the most efficient lignocellulose degradation ability in nature, and is one of the idea microorganisms to produce biofuels and chemicals from lignocellulose. The high efficiency of C. thermocellum is depend on its multi-enzyme complex, cellulosome. The cellulosome is capable of converting a wide variety of plant cell wall polysaccharides directly into soluble sugars. It has been reported that the components and structure of cellulosome is regulated by the extracellular substrates, probably through several couples of sigma and anti-sigma factors. Understanding the signal pathways through sigma factors and their corresponding anti-sigma factors is helpful to rational design of cellulosme for developing a more efficient system to degrade lignocelluloses. Secondary structure prediction and transmembrane helix prediction indicated that the anti-sigma factors for cellulosome regulation in C. thermocellum have an extracellular carbohydrate-binding domain, a disorder area across cell wall, a periplasmic domain, a transmembrane helix and an intracellular domain.This thesis studied an anti-sigma factor RsgI2 encoded by Cth₀267 of C. thermocellum ATCC 27405 by combining molecular biology, protein biochemistry, and nuclear magnetic resonance techniques.?1? Expression, purification and NMR structure determination of the intracellular domain of the anti-sigma factor RsgI2 in C. thermocellumThe intracellular domain of RsgI2 was cloned into vector pET-30a?+? and overexpressed by Escherichia coli BL21?DE3?. The protein product contains an additional C-terminal His-tag to facilitate the protein purification. The recombinant RsgI2 intracellular domain was purified using Ni+ affinity chromatography and anion exchange chromatography.15N/13C-labeled proteins were obtained by cell growth in M9 media containing 15NH4Cl and [13C]-glucose as the sole nitrogen and carbon sources, respectively. After NMR experiments were performed using the labelled proteins, all NMR spectra were processed and analyzed, and the chemical shift of main chain and side chain atoms were assigned. Finaly, we obtained the solution structure by structure calculation. The intracellular domain of Rsgl2 represents an OB-fold type structure with four ? strands. OB-fold structures widely exsit in proteins which bind to nucleic acid, polysaccharide or special ligand. RsgI2 intracellular domain has a solvent-exposed hydrophobic surface, which is probably important to sigma factor binding.?2? Expression, purification and NMR structure determination of the periplasmic domain of the anti-sigma factor RsgI2 in C. thermocellumThe periplasmic domain of RsgI2 was cloned into vector pET-30a?+? and overexpressed by E. coli Transetta?DE3?. The protein product contains an additional C-terminal His-tag to facilitate the protein purification. The protein was purified using Ni2+ affinity chromatography and gel filtration chromatography.15N/13C-labeled proteins were obtained by cell growth in M9 media containing 15NH4Cl and [13C]-glucose as the sole nitrogen and carbon sources, respectively, and were used for NMR experiments. All the NMR spectra were processed and analyzed, and the chemical shift of main chain and side chain atoms were assigned. Finaly, we obtained the solution structure by structure calculation. The periplasmic domain represents an ?/?/? sandwich stucture with seven a helixs and six ? strands. The topology type is novel among known ptotein structures. The two structures provide the structural foundation to the research of signal transduction pathways of sigma factors and their corresponding anti-sigma factors.?3? Preliminary study of signal transduction pathways through sigma factors and their corresponding anti-sigma factorsThe sigma factor SigI2 corresponding to RsgI2 was expressed and purified, electrophoretic mobility shift assays ?EMSA? were performed with SigI2 and promotor regions of cellulosome genes for studying the promoter recognition mechanism. As a result, Sigl2 can bind to promoter of CelS and Sigll, but not to SigA promoter, which proved that sigma factors recognize cellulosome promoters.We tried to use native-PAGE, gel filtration chromatography and NMR titration to study the interaction between RsgI2 intracellular domain and Sigl2. SigI2 can not enter the gel in the native-PAGE, so we could not use native-PAGE to verify the interaction between them. Gel filtration chromatography and NMR titration experiments proved the interaction between the two proteins, which indicated that sigma factors and their corresponding anti-sigma factors can recognize each other. These results provide the basis to further mutagenesis analysis and structure-function analysis study to elucidate the signal pathways mechanism of the sigma-anti-sigma factors.