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
<p><b>OBJECTIVE</b>To investigate the mRNA expression of transferrin receptor 1 (TfR1) and iron regulatory protein 1 (IRP1) in the full-term placenta from different maternal iron status, and explore the mechanism of placental iron transport and regulation.</p><p><b>METHODS</b>The mRNA level of TfR1 and IRP1 in full-term placentae was detected by reverse transcription polymerase chain reaction (RT-PCR) in normal group (N), iron deficiency group (ID) and iron deficiency anemia group (IDA).</p><p><b>RESULTS</b>(1) The expression of TfR1 mRNA in N group was 0.4813 +/- 0.1891, in ID group was 0. 6647 +/- 0.2788, and in IDA group was 0.9767 +/- 0.2858. There was significant difference between IDA group and N group or ID group (t = 0.002, P < 0.01 or t = 0.028, P < 0.05), and was no difference between ID group and N group (t = 0.117, P > 0.05). (2) The expression of IRP1 mRNA in N group was 0.2616 +/- 0.0785, in ID group was 0.3696 +/- 0.1801, and in IDA group was 0.3971 +/- 0.0902 and was no difference among the three groups (F = 1.845, P = 0.179).</p><p><b>CONCLUSIONS</b>The expression of TfR1 mRNA is increased when maternal iron deficiency progressed while there is no change in the expression of IRP1 mRNA in the placentae of TfR1 mRNA indicated that IRP1 takes part in the regulation of placenta iron transport.</p>