Purification And Function Of Bacterioferritin In Anammox Bacteria

Anaerobic ammonia oxidizing bacteria(hereafter referred to as “An AOB”),under the condition of no carbon source or oxygen,uses nitrite as an oxidizing agent to convert ammonium salts into nitrogen,from which it gains energy.Mature An AOB contain a lot of cytochrome and most of them are in anammoxosome.And Fe,as an important component,plays an important role in the function of An AOB.However,the research on the function mechanism of Fe in An AOB is still very lacking.Previous studies have found that there are some Fe-rich nanoparticles in An AOB,which may be an Fe storage protein that plays a key role in the Fe metabolism system of bacteria.This thesis,taking the bacterioferritin discovered in An AOB as main object,explored the expression and function of bacterioferritin and its regulatory mechanism in bodies,which provided theoretical support for iron regulation system in An AOB?(1)Objective to optimize the expression and purification conditions of ferritin in vitro.First,based on the ferritin Q1Q5F8 discovered in An AOB in previous studies,an expression system of ferritin in Escherichia Coli was constructed by using biological methods.Subsequently,the temperature,inductor concentration and buffer concentration were optimal in the process of culture of Ks Bfr.culture conditions for inducing the expression of the target protein and the protein purification conditions were determined to finally obtain the spherical ferritin with high purity and complete assembly,with a diameter of about 15-18 nm.(2)Verify the function of ksbfr.The growth of engineering bacteria containing the recombinant protein with different gradient concentrations of Fe SO4 was observed.The results showed that the growth of engineering bacteria was inhibited when the concentration of Fe SO4 was more than 5 mm.The activity of engineering bacteria was higher than that of wild Escherichia Coli under the condition of 2.5 mm Fe SO4,and the survival rate was about 10% higher than that of wild bacteria.This indicated that the iron storage protein of anammoxides can also be expressed in E.coli and play a role in iron storage.The purified protein monomer can also convert Fe(?)into Fe(?),and found that there have active ferrioxidase center.In addition,transcriptome analysis indicated that exogenously implanted ferritin may also be affected by Fur,the Fe regulatory protein within the expression system.It improves the expression of ferritin in engineered bacteria,speeds up the expression of genes involved in Fe transport and metabolism,and raises the upper limit of the utilization of ferrous ions,allowing protection from the harmful effects of too much Fe in the body.(3)Bioinformatics of ksbfr.Through bioinformatics technology,the physical propertie and structural characteristics of ferritin in An AOB were further grasped by software.The results show that ksbfr is a kind of hydrophobic protein with strong stability.The spherical structure of its 24 polymer was obtained by homology modeling.Through genetic analysis,it was found that it was closer to Pseudomonas aeruginosa.By analyzing the functional active site and conserved structure of the protein,it was found that there were three functional structures: ferrous oxidation center,heme binding site and Fe oxidation channel.