Directed Evolution Of Lead Binding Protein PbrR With Dual Selection System

The pbr operon in Cupriavidus metallidurans CH34 is responsible for the uptake,translocation and enrichment of lead ions,in which lead binding protein PbrR regulates the expression of the entire operon and specifically binds lead ions.However,natural protein is easily interfered by other metal ions such as zinc ions,which limits the application potential of protein to some extent.In order to reduce the interference of zinc ions,this study used directed evolution technology combined with the dual selection system to achieve the specific enhancement of lead binding protein PbrR,and to develop a lead binding protein with higher specific binding to lead ions.Dual selection system consists of the positive screening marker gene amp and the reverse screening marker gene sacB,which are regulated by the expression of PbrR.The optimization results of the screening conditions indicated that the cells with selection plasmid grew more rapidly with the condition of ampicillin 100?g/mL and lead ions 50?M due to more expression of positive selection markers,which can be used as ON screening conditions.When cultured with sucrose 10% and zinc ions 50 ?M,the cells initially showed significant growth inhibition,which can be used as an optimal condition for OFF screening.In this condition,reverse selection marker was expressed because zinc ion also showed the binding with PbrR.The key gene pbrR was amplified by error-prone PCR used for the introduction of random mutations,and then combined with repeated directional screening.Finally,mutant strains M1 and M2 with rapid growth under ON and OFF screening conditions were obtained.Compared with the wild type,the mutant strains grew faster and binding ability of lead ion was stronger of lead ion,and the growth value increased 1.8 times.The growth of wild type was inhibited on OFF screening,while M1 and M2 grew rapidly,so the binding ability of zinc ion was weakened.Homology modeling with a known structural protein PbrR691 establishes a structural model of homodimer protein PbrR,preliminary analysis of the relationship between protein and metal ion binding ability.Mutation C134 R of M1 was located on the C-terminal metal-binding loop region,which may lead to additional enhancement of cadmium ion binding.Double mutations D64 A and L68 S of M2 were located on the ?-helix ?4 near the loop region of C79.Amino acid mutations near the metal binding domain of the nearby dimeric protein may cause subtle force changes and spatial changes,leading to reduced binding capacity of zinc ions.