Preliminary Crystallographic Studies Of TteRRF And AmCBL1, And Crystal Growth Study Of ATPase From A Thermophilic Bacterium ATCC27502

Ribosome recycling factor (RFF) is a kind of protein that breaks down the post-termination complex in the protein synthesis process divided into four steps that are initialization, elongation, termination and dissembling of post-termination complex. A series of TteRRF C-terminal mutants and other hemophilic bacterium show different characters in rescuing temperature sensitive phenotype E. coli strain (frrts phenotype) at non-permission temperature, and in order to illustrate these phenomena from molecular level, determination of its structure become urgent. TteRRF was constructed in the vector pET-DB, overexpressed in E.coli. TteRRF purified through three step chromatography (Ni affinity, ion exchange, and size exclusion chromatography) was crystallized using reduction methylation method. A complete data from a crystal has been collected at 2.8 A and the crystal belongs to the space group P6122/P6522,with unit-cell parameter a=103.264A,b=103.264A, c=89.173A, a=90.000,β=90.000,γ=120.000.ATPase (F0F1-ATPase) existing in various biomenbranes catalyze the synthesis of ATP droved by H+ Electric chemical gradient and transport the H+ by hydrolysis the ATP. F0F1-ATPase in prokaryotes consisting of two part s-F1 (α3(33γδε) in the cytoplasm and F0 (ab2c) in the membrane is a transmembrane super-molecular complex. Although structures of most of F0F1-ATPase subunits except a subunit and c-terminal of b subunit have been solved, there is no structure report about the F0F1-ATPase as a whole enzyme to this day. F0F1-ATPase from the thermophilic bacterium ATCC27502 was chosen as study model. The whole molecular was purified natively, SDS-PAGE indicating 70% purity, and have the ATP hydrolysis ability. Dynamic light scattering (DLS) shows that it is monodispersity which suit for screening crystal. We finally obtained the microcrystal using hanging drop method, providing the foundation for the next step of optimization. CBL1(Calcineurin B-like protein 1) is a calcium sensor that transmits the calcium signal to its target CIPKs (CBL-interaction protein kinase).CBL1 and CIPKs play a crucial role in plant response to the environmental stresses such as low K+, osmotic, salt, cold and drought. The wild type and serious of mutant AmCBLΔ17 genes were inserted into the vector pET-22b(+), and then the E.coli strain BL21 (DE3) was transformed. The overexpressed protein was purified using Ni affinity chromatography, DEAE ion exchange chromatography and size exclusion chromatography. The purified protein was screened for crystallization condition. Finally, the mutant form AmCBL1Δ17-123 gave rise to the best diffraction quality crystals. Diffraction data sets have been collected to 2.9 A resolution. The crystals belong to space group P21212, with unit cell parameters a=99.87 A, b=114.42 A, c= 63.80A,α=β=γ=90.00.