Expression, Purification And Crystal Structure Of Ectoine Hydroxylase

Ectoine and 5-hydroxyectoine have wide use and application prospect in fine chemical industry, biological medicine, biological manufacturing and production, environmental protection and other fields. Ectoine hydroxylase can catalyse the substrate ectoine to form hydroxyectoine with the cofactor Fe2+ and co substrate 2-oxoglutarate,so it is the key enzyme of microbial fermentation for hydroxyectoine. Protein crystallography utilizes protein crystallization and X-ray diffraction techniques to determine the structure of proteins, it can help us understand the structure of proteins at atomic scale, determine how the enzyme functions and clarify the key residues participating in a certain reaction process, and these informations would help us in modifying and transforming of key enzymes.In this study, ectoine hydroxylase from Bacillus pseudofirmus OF4 was set as the research object exploring its expression, purification and crystallization an so on.Using the principles of genetic engineering, the target gene was successfully constructed into the expression vector pET-22b(+) and overexpressed in Escherichia coli strain BL21 (DE3). With the His6-tag, the recombinant protein with the purity above 95% was obtained after isolation and purification by Ni2+-chelating affinity chromatography,SuperdexTM 200 10/300 GL gel filtration chromatography and Resource Q anion exchange chromatography. Using the crystallization commercial kits, the target protein was crystallized by hanging-drop and sitting-drop vapour-diffusion methods for screening. After optimizing the crystallization conditions,crystal samples with high resolution were obtained by sitting-drop vapour-diffusion method with a solution consisting of 200 mmol/L Magnesium chloride hexahydrate,100 mmol/L BIS-TRIS pH 6.5,25% (w/v) Polyethylene glycol 3,350 and the purified protein concentration of 6.5 mg/ml. The ideal crystal diffracts to 2.40 A resolution and belongs to triclinic space group PI, with unit-cell parameters a= 45.183 A, b= 58.871 A, c= 68.812 A,α=77.478°, β=86.034°,γ=66.966°. With the molecular-replacement (MR) method, the three-dimensional structure of the protein is successfully determined. In an asymmetry unit,there are two protein molecular,and a monomer cosists nine a-helixes and nine (3-sheets,which are linked by several P-turns and loops. Obtaining of the three-dimensional structure of EctD will further provides important guiding information for understanding its structures and biological function.Furthermore, the complex crystallizations of EctD-2-oxoglutarate and EctD- ectoine are in progress. Specifically,the X-ray diffraction resolution of EctD and 2-oxoglutarate complex crystal reaches 3.50 A, and the complex crystal needs to be further optimized.