Increase of Solubility of Foreign Proteins in Escherichia coli by Coproduction of the Bacterial Thioredoxin

Yasukawa, Takashi; Kanei-Ishii, Chie; Maekawa, Tomoko; Fujimoto, Jiro; Yamamoto, Tadashi; Ishii, Shunsuke

doi:10.1074/jbc.270.43.25328

Cited by 284 publications

(164 citation statements)

References 26 publications

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“…Phosphorylated GST-fusion proteins were produced as recommended by the manufacturer. The corresponding nonphosphorylated molecular species were isolated from E. coli strain BL21(DE3)-pT-Trx (Trx) (Yasukawa et al, 1995). Puri®ed GST-fusion proteins were bound for 1 h at 48C to glutathione (GT) agarose beads (40 ml slurry; Pharmacia) suspended in FMIP/Fms binding bu er (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.01% Tween20, 1% Trasylol, 20 mg/ml BSA and 200 mM sodium orthovanadate).…”

Section: Binding Of Cellular Proteins To Gst-fmip Fusion Proteinsmentioning

confidence: 99%

FMIP, a novel Fms-interacting protein, affects granulocyte/macrophage differentiation

et al. 1999

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Hematopoietic cell growth, di erentiation, and commitment to a restricted lineage are guided by a set of cytokines acting exclusively on cells expressing the corresponding cytokine receptor. The macrophage colony stimulating factor (M-CSF, also termed CSF-1) and its cognate receptor, the tyrosine kinase c-Fms, are essential for monocyte and macrophage development. The underlying molecular mechanism, however, is poorly understood. Here we identi®ed a novel Fms-interacting protein (FMIP, MW 78 kDa) which binds transiently via its Nterminal 144 residues to the cytoplasmic domain of activated Fms-molecules. Binding of FMIP was paralleled by rapid tyrosine phosphorylation within the binding domain which drastically reduced its ability to associate with Fms. Binding was speci®c as evidenced by co-immunoprecipitation and association with recombinant GST-Fms fusion proteins. No binding was observed with the tyrosine phosphorylated cytoplasmic domains of c-Kit, TrkA, c-Met, and the insulin receptor. The role of FMIP in hematopoietic di erentiation was studied in the bipotential myeloid progenitor cell line, FDC-P1Mac11. Overexpression of FMIP prevented M-CSF induced macrophage di erentiation. Instead, cells di erentiated into granulocytes. Our data suggest that the level of FMIP expression could form a threshold that decides about di erentiation either into macrophages or into granulocytes.

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Section: Binding Of Cellular Proteins To Gst-fmip Fusion Proteinsmentioning

confidence: 99%

FMIP, a novel Fms-interacting protein, affects granulocyte/macrophage differentiation

et al. 1999

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“…However, we were not successful in these attempts, since most of the protein was produced in an insoluble form and we were unable to recover it by solubilization and renaturation. In an attempt to solve this problem, we decided to use folding catalysts to help expression of the protein in a soluble form, an approach that has been used by other investigators with success (20)(21)(22)(23)(24). The simultaneous expression of the bacterial chaperonins GroES/GroEL using the pT-GroE plasmid highly increased the expression of soluble IRP1, as shown in Figure 1.…”

Section: Resultsmentioning

confidence: 99%

“…Soluble protein recovery from these inclusion bodies is a difficult and often unsuccessful task. The use of molecular chaperonins to facilitate soluble protein expression has been adopted in some systems with success (20)(21)(22)(23)(24). Chaperonins are ubiquitous proteins that have an important role in the folding of newly synthesized proteins in vivo.…”

Section: Introductionmentioning

confidence: 99%

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

Carvalho

Meneghini

2008

Braz J Med Biol Res

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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.

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“…Expression and preparation of GST-CBP, GST-GATA-1, GST-c-Myb fusion proteins, GST alone, full-length c-Myb, or Dc-Myb were carried out as described (Nomura et al, 1993;Yasukawa et al, 1995). In vitro translation was carried out using the TNT reticulocyte lysate system (Promega).…”

Section: Protein Expression and In Vitro-binding Analysis With Gst Fumentioning

confidence: 99%

Inhibitory interaction of c-Myb and GATA-1 via transcriptional co-activator CBP

et al. 2000

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Gene targeting experiments have revealed that transcription factors such as c-Myb and GATA-1 play crucial roles during hematopoietic di erentiation. c-Myb is necessary in the immature cells of almost every hematopoietic lineage and GATA-1 is essential for the development of the erythroid lineage. In addition, CREB-binding protein (CBP) acts as a transcriptional adapter for various transcription factors, including cMyb and GATA-1. In this paper, we show that the transcription factors c-Myb and GATA-1 each inhibit the transcriptional activity of the other and that any possible bipartite complexes c-Myb, GATA-1, and CBP could be formed, but the tripartite complex was hardly formed. The exclusive binding of GATA-1 and c-Myb to CBP is probably the molecular basis for the mutual inhibition of their transcriptional activity. Our data suggest that cross-talk between these three factors might be important for hematopoietic di erentiation and that CBP functions as a key molecule during the process.

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Increase of Solubility of Foreign Proteins in Escherichia coli by Coproduction of the Bacterial Thioredoxin

Cited by 284 publications

References 26 publications

FMIP, a novel Fms-interacting protein, affects granulocyte/macrophage differentiation

FMIP, a novel Fms-interacting protein, affects granulocyte/macrophage differentiation

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

Inhibitory interaction of c-Myb and GATA-1 via transcriptional co-activator CBP

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