ABSTRACT
Objective To investigate the relationship between the expressions of iron transport related proteins and the dysregulation of iron homeostasis in the spinal cord of amyotrophic lateral sclerosis (ALS) transgenic mice. Methods The hSOD1
ABSTRACT
Iron is an essential metal for all living organisms. However, iron homeostasis needs to be tightly controlled since iron can mediate the production of reactive oxygen species, which can damage cell components and compromise the integrity and/or cause DNA mutations, ultimately leading to cancer. In eukaryotes, iron-regulatory protein 1 (IRP1) plays a central role in the control of intracellular iron homeostasis. This occurs by interaction of IRP1 with iron-responsive element regions at 5' of ferritin mRNA and 3' of transferrin mRNA which, respectively, represses translation and increases mRNA stability. We have expressed IRP1 using the plasmid pT7-His-hIRP1, which codifies for human IRP1 attached to an NH2-terminal 6-His tag. IRP1 was expressed in Escherichia coli using the strategy of co-expressing chaperonins GroES and GroEL, in order to circumvent inclusion body formation and increase the yield of soluble protein. The protein co-expressed with these chaperonins was obtained mostly in the soluble form, which greatly increased the efficiency of protein purification. Metal affinity and FPLC ion exchange chromatography were used in order to obtain highly purified IRP1. Purified protein was biologically active, as assessed by electrophoretic mobility shift assay, and could be converted to the cytoplasmic aconitase form. These results corroborate previous studies, which suggest the use of folding catalysts as a powerful strategy to increase protein solubility when expressing heterologous proteins in E. coli.
Subject(s)
Humans , Chaperonin 10 , Chaperonin 60 , Escherichia coli/metabolism , Iron Regulatory Protein 1/metabolism , Chaperonin 10 , Chaperonin 60 , Chromatography, Ion Exchange , Electrophoretic Mobility Shift Assay , Escherichia coli/genetics , Gene Expression , Iron Regulatory Protein 1/isolation & purification , RNA-Binding Proteins , SolubilityABSTRACT
Objective To investigate the expressions of transferrin receptor (TfR) mRNA, ferritin(Fn) mRNA and iron regulatory proteinl (IRP-1) mRNA in the placentas complicated with fetal iron deficiency(ID). Methods Depending on the cord SF,the subjects were divided to fetal ID group and fetal iron sufficient( IS) group. TfR mRNA, Fn mRNA and IRP - 1 mRNA in placentas were measured by RT - PCR. Results 1. The expression of TfR mRNA in ID group was 1.10 ? 0. 26, it was significantly higher than that in IS group (t=0.028 P0.05) ;4. TfR mRNA and Fn mRNA correlated with fetal IS respectively. Conclusion Fetal ID induces highly expressed TfR mRNA and lower level of Fn mRNA to furthest satisfy the iron need for fetus.