Expression, Purification And Researches Of The Function Of Ferric-ion Binding Protein, Hepcidin And MnSOD

1. Ferric-ion binding proteinFbp, which contains 309 residues, is expressed by Gram-negative bacteria and located in cytoplasma to transport Fe3+ across the cytoplasm. It is a major protein of pathogenic bacteria to sequester ferric-ion. Fbp binds an iron by one histidine (His9), one glutamate (Glu57) and two tyrosines (Tyrl95, Tyr196) residues, with a phosphate as a synergistic anion and a water as the sixth ligand. It was found that the two tyrosine motif is important for the protein to bind Fe3+, and the synergistic anion also plays an important role for the metal binding.To investigate the effect of the two tyrosine motif on Fbp binding to Fe3+, we expressed and purified Fbp mutants Y195F, Y196F and Y195F/Y196F, and researched the reaction between Fe(NTA) and Y195F by UV/vis spectroscopy. A red shift of the characteristic absoption peak from 481 nm to 468 nm was found, which indicated that Fe(NTA) bound to Y195F to form a ternary complex Fe-Y195F-NTA. There is about 0.5 eq. of Fe3+ in per mol of the protein mutant, and the apparent Fe3+-Fbp binding constant is ca.3.1×104L·mol-1(in 10 mmol·L-1 Hepes buffer, pH 7.4,298K).It was found that Fbp also exhibited a function to catalyze hydrolysis of phosphate ester in our Lab, which was also reported in some literatures. In order to investigate the enzymic mechanism of the hydrolysis reaction, we added PPi into apo-Fbp and its mutants at different pH(in 10 mmol·L-1 Hepes buffer,298K). The different rate constants of reaction we calculated were 1.05 mmol·L-1·h-1.1.11 mmol·L-1·h-1,1.22×10-2 mmol·L-1·h-1 at pH6.5,7.5 and 8.5 respectively, and similar with the value of apo-Fbp. But the value of Y195F was 10 folds lower than apo-Fbp-s. At the same time we incubated PPi with apo-Fbp and its mutants for different time (10mmol·L-1 Hepes buffer, pH 7.4,293K), respectively, and hybrided the reaction samples to phosphate-tyrosine antibody (Dotblot). The result showed that apo-Fbp, Y195F and Y196F, but not the bimutant Y195F/Y196F, bound with phosphate. This confirmed that Fbp catalyzing the hydrolysis of PPi conformed to the mechanism of critical residue tyrosine 195 phosphorylation on the protein.2. HepcidinHepcidin (HEP), a 25-amino-acid and cysteine-rich peptide, is secreted in hepatocyte to regulate iron homeostasis. Expression of HEP is positively regulated by iron level and inflammation and negatively regulated by anemia and hypoxia. Hepcidin is related to the iron overload diseases, such as hemochromatosis, neurodegenerative diseases.Hepcidin-25 cDNA was cloned and then subcloned into vector pMal-C2X. The constructed plasmid was transfected ino E. coli to express fusion protein MBP-hepcidin. A soluble protein with molecular-wight 46000Da was purified using affinity column. After disgested with suitable digestive enzymes, hepcidin-25 monomer could be obtained, which can be used to investigate its roles in controlling iron homeostasis.3. Manganese Superoxide dismutaseManganese superoxide dismutase (MnSOD), which expressed in mitochondria of eucaryote cell, is the primary antioxidant enzyme to protect cells from oxidative stress by catalyzing dismutation of superoxide (O2-).In this study, MBP-MnSOD plasmid was transferred into E. coli Rossetta DE3 plus, which was then induced with IPTG to express the soluble fusion protein MBP-MnSOD. Purified MBP-MnSOD was obtained using amylose affinity column and steric exclusion chromatography. This work creates a foundation for the researches on the metabolism and fuction of Mn, and MnSOD as well.