Study Of Biochemical Activity And Molecular Mechanisms Of Bacterial Ferritin

Ferritin is a protein of iron oxidation,storage and mineralization proteins found throughout the animal,plant,and microbial kingdoms.In recent years,with the deep understanding of ferritin structure,ferritin has attracted attention in the bioengineering and biomedical fields based on its structural and magnetic properties.In this paper,ferritin derived from Escherichia coli and Pyrococcus furiosus was used in the research to study ferritin expression,magnetic formation and the mechanism of Ferroxidase center.In this study,the protein expression system of Escherichia coli and Pyrococcus furiosus ferritin was constructed.The inducer concentration and iron ion concentration of normal expression of ferritin were determined.Secondly,we determined the assembly state of ferritin in vitro with Native gel,and optimized the concentration of separation gel and electrophoresis temperature.At the same time,we determined that the ultrasonic disruption method can obtain a large amount of soluble ferritin by comparing the different bacterial disruption methods.Compared with the freeze-thaw method and the mechanical grinding method,this method is easy to operate and to the maximum extent avoid the impact of iron protein caused by the complex operation.For the purpose of developing ferritin magnetic separation tags,we studied the magnetic properties of ferritin.A separation device for an iron powder-packed column capable of rapidly separating magnetic ferritin was determined.Based on this device,we identified the types of buffers that can minimize the non-specific adsorption of ferritin,and further explored other influencing factors in the magnetic separation process.At the same time,in view of the mechanism of iron oxidase ferritin based on the micro-isothermal titration calorimetry,we initially explored the titration curves of purified ferritin using different concentrations of ferrous sulfate,it was found that the mutant ferritin has a high affinity for iron ions.Although the titration conditions of this experiment require us to further explore and optimize,this method also provides new ideas for the study of ferritin magnetic mechanism.In summary,the phenomenon of magnetic formation of ferritin and its related mechanism of action in bacterial ferritin have been studied from various aspects.The optimum conditions for the expression of ferritin were determined,and a variety of factors influencing the ferritin's magnetic formation and magnetic separation efficiency were thoroughly studied.The research will promote the application of ferritin in bioengineering,drug delivery,biomedicine,and nanomaterials.