Increased expression and purification of soluble iron-regulatory protein 1 from Escherichia coli co-expressing chaperonins GroES and GroEL

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 NH 2 -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.

Repression of ferritin light chain translation by human eIF3

“…IRP1 was purified based on the protocols in Carvalho and Meneghini (2008), Basilion et al (1994) with modifications. The IRP1-encoding 2B-T plasmid was transformed into chemically-competent BL21 Rosetta pLysS E. coli , using heat shock at 42 °C, and grown on Ampicillin plates.…”

Use of folding modulators to improve heterologous protein production in Escherichia coli

“…Overproduction of GroESL has proven a highly productive approach to overcoming polypeptide folding problems in E. coli , allowing the soluble production of many recombinant proteins which are otherwise produced exclusively or almost exclusively in inclusion bodies. These include proteins as diverse as human thromboxane synthase [ 60 ], nicotinoprotein formaldehyde dismutase from Pseudomonas putida F61 [ 61 ], human oxygen-regulated protein ORP150 and human lysozyme [ 17 ], a human iron-regulatory protein [ 62 ], a putative bacterial dehydratase [ 63 ], β-glucosidases from Cellovibrio gilvus and Agrobacterium tumefaciens [ 64 ], murine c-Myb, cAMP response element-binding protein 1, p53 tumour suppresor gene product, Xenopus mos proto-oncogene product [ 65 ], bacterial magnesium transporter CorA [ 66 ] and triazine hydrolase from Arthrobacter aurescens TC1 [ 67 ]. A sample of proteins whose total or functional yield in the E. coli cytoplasm is merely increased upon GroESL overproduction, meanwhile, can be found in Table 1 [ 19 , 21 , 36 , 39 , 43 , 58 , 61 , 64 , 68 - 101 ].…”

Co-expression of chaperones from P. furiosus enhanced the soluble expression of the recombinant hyperthermophilic α-amylase in E. coli

“…For examples, co-expression with the trigger factor (TF) alone was sufficient to prevent the aggregation of mouse endostatin, while overexpression of TF together with GroEL-GroES was more effective for human ORP150 and lysozyme (Nishihara et al 2000). Overexpression of GroEL-GroES was also able to greatly increase the yield of soluble ironregulatory protein 1 (Carvalho and Meneghini 2008) and to enhance the soluble expression of human interferon-γ (Yan et al 2012). de Marco (2007) co-expressed eight combinations of E. coli molecular chaperones with a target protein in order to improve target protein's soluble yield.…”