The Expression, Purification, Structure And Function Of Cadr,a Cadmium Regulatory Protein

A chromosomal fragment encoding cadmium resistance was cloned from Pseudomonas putida06909, a kind of rhizosphere bacterium. It revealed two divergently transcribed genes, cadA and cadR by sequence analysis. The CadR is a MerR family metalloregulatory protein according to homologous sequence alignment which consists of147amino acid residues including five cysteines with16648Da molecular weight.In this paper, the CadR-pET30a plasmid that contains CadR gene with optimized codon by total gene synthesis is constructed. And the CadR protein can be efficiently expressed in E.coli BL21strain. Through three step purification by QFF column, MonoQ column and Gel Filtration column, the CadR protein are ready to setup drop in crystal growing experiment and biochemical assay. The EDTA competition experiment result shows that the protein can specially bind with Cd2+and conditional equilibrium constant of Cd-CadR complex is about2.5×1013at pH7.4. Subsequently, the DNA footprinting analysis and EMSA assay results disclose the accurate binding area which the CadR interact with promoter DNA. So the expressed recombinant CadR proteins have both good N-terminal protein-DNA interaction activity and C-terminal metal binding activity.We also fortunately optimize several the CadR protein crystallization condition by Hampton Research Kits. The crystal can be diffracted to2.1A resolution at U-17beam station in Shanghai Synchrotron Radiation Facility. The crystal structure of TC21-CadR shows that the CadR N-terminal DNA binding region has helix-turn-helix structure just like other MerR family protein used. The CadR C-terminal metal binding site consists of3cysteines (C77, C112’, C119’) from different monomer. The D74GD75G-CadR mutant ITC experiment results show that the two aspartic acids (D74, D75) in the metal binding center area have no apparent coordination activities with Cd2+binding.