Renaturation and Purification of Biologically Active Recombinant Human Macrophage Colony-Stimulating Factor Expressed in E. Coli

Halenbeck, Robert; Kawasaki, Ernest S.; Wrin, Joe; Koths, Kirston

doi:10.1038/nbt0789-710

Cited by 91 publications

(41 citation statements)

References 25 publications

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“…Some of the monomeric species migrated with apparent molecular weights significantly smaller than that of the reduced, denatured M-CSF monomer, suggesting a compact, folded structure. These intermediate forms were also reported by Halenbeck et al (1989) for a longer version of M-CSF. In addition, these "folded monomers" moved with a decreased retention time on reversed-phase HPLC when compared to the reduced monomer or the native dimer.…”

Section: Discussionsupporting

confidence: 75%

A study of intermediates involved in the folding pathway for recombinant human macrophage colony‐stimulating factor (M‐CSF): Evidence for two distinct folding pathways

et al. 1993

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The folding pathway for a 150-amino acid recombinant form of the dimeric cytokine human macrophage colony-stimulating factor (M-CSF) has been studied. All 14 cysteine residues in the biologically active homodimer are involved in disulfide linkages. The structural characteristics of folding intermediates blocked with iodoacetamide reveal a rapid formation of a small amount of a nonnative dimeric intermediate species followed by a slow progression via both monomeric and dimeric intermediates to the native dimer. The transition from monomer to fully folded dimer is complete within 25 h at room temperature at pH 9.0. The blocked intermediates are stable under conditions of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and thus represent various dimeric and folded monomeric species of the protein with different numbers of disulfide bridges. Peptide mapping and electrospray ionization mass spectrometry revealed that a folded monomeric species of M-CSF contained three of the four native disulfide bridges, and this folded monomer also showed some biological activity in a cell-based assay. The results presented here strongly suggest that M-CSF can fold via two different pathways, one involving monomeric intermediates and another involving only dimeric intermediates.

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Section: Discussionsupporting

confidence: 75%

A study of intermediates involved in the folding pathway for recombinant human macrophage colony‐stimulating factor (M‐CSF): Evidence for two distinct folding pathways

et al. 1993

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“…The cells were then washed three times in R10 in order to remove IL-3 before plating in Difco (Detroit, USA) soft agar at concentrations of 1610 6 , 1610 5 , and 1610 4 cells/ml in R10 with or without 5000 U/ml of recombinant human M-CSF with a speci®c activity of 5610 7 units per mg (Ladner et al, 1987;Halenbeck et al, 1989) provided by Chiron (Emeryville, CA, USA). Alternatively 2610 5 washed cells were seeded into 4 mls of R10 with or without 5000 U/ml M-CSF in six well plates.…”

Section: Transformation Assaysmentioning

confidence: 99%

Cell specific transformation by c-fms activating loop mutations is attributable to constitutive receptor degradation

et al. 1999

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Expression of a receptor for human macrophage-colony stimulating factor (M-CSF or CSF-1), containing a point mutation which changes an aspartate to a valine at position 802 of the activating loop of the kinase domain, potently transforms the haemopoietic cell line FDC-P1 yet prevents Rat-2 ®broblast transformation. In order to understand this apparent paradox, aspartate 802 was changed by cassette mutagenesis to each of the other 19 amino acids. All hydrophobic amino acid substitutions were transforming when tested in FDC-P1 cells yet inactivating when tested in Rat-2 ®broblasts. These same amino acid substitutions also activated receptor degradation, strongly suggesting a causal relationship between receptor degradation and inactivation in ®broblasts. Point mutations or small deletions of Y708 within the kinase insert region of the mutant D802V receptor partly inhibited receptor degradation. The more stable D802V receptor derivatives were able to transform both FDC-P1 cells and Rat-2 ®broblasts, so establishing that the cell speci®c e ect of the c-fmsD802V activating loop mutation is attributable to receptor degradation which accompanies kinase activation and prevents the transformation of Rat-2 but not of FDC-P1 cells.

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“…NIH 86-23, 1985). Four normal and four mutant rats were treated with highly purified recombinant human CSF-1 (Ladner et al, 1987;Halenbeck et al, 1989), which was generously provided by Chiron Corporation. Administration of the growth factor was by subcutaneous injection of 10 6 units every 48 hours (Ralph et al, 1986), beginning at birth for a period of 2 weeks.…”

Section: A Source and Treatment Of Animalsmentioning

confidence: 99%

Growth Hormone Receptor and Insulin-like Growth Factor-I Immunoreactivity in Osteoclast-like Cells During Tooth Eruption in the Toothless (Osteopetrotic) Rat Following Treatment with Colony-Stimulating Factor-1

Symons

Weerakoon

Marks

2003

Crit Rev Eukaryot Gene Expr

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ABSTRACT:In the toothless (tl/tl) osteopetrotic rat, teeth form but fail to erupt. Treatment of tl/tl rats with colony-stimulating factor-1 (CSF-1) activates bone resorption by osteoclasts, permits tooth eruption, and upregulates the immunoreactivity of bone marrow mononuclear cells to growth hormone receptor (GHr) and insulinlike growth factor (IGF)-I. This study examined the distribution of tartrate-resistant acid phosphatase (TRAP) and immunoreactivity for GHr and IGF-I in osteoclast-like cells located on the alveolar bone margin, adjacent to the lower first molar crown, in 14-day-old normal and tl/tl rats, following treatment with CSF-1. Osteoclast-like cells demonstrated a positive reaction for TRAP, GHr, and IGF-I in all groups. However, in tl/tl tissue, osteoclast-like cells were generally negative for GHr. There was no significant difference in the total number of TRAP-, GHr-, and IGF-I-positive osteoclast-like cells on the adjacent bone margin in normal, normal treated with CSF-1, and tl/tl rats. CSF-1 treatment of the tl/tl rat significantly increased the total number of osteoclast-like cells, which were positive for TRAP (p < 0.001), GHr (p < 0.05) and IGF-I (p < 0.01).

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Renaturation and Purification of Biologically Active Recombinant Human Macrophage Colony-Stimulating Factor Expressed in E. Coli

Cited by 91 publications

References 25 publications

A study of intermediates involved in the folding pathway for recombinant human macrophage colony‐stimulating factor (M‐CSF): Evidence for two distinct folding pathways

A study of intermediates involved in the folding pathway for recombinant human macrophage colony‐stimulating factor (M‐CSF): Evidence for two distinct folding pathways

Cell specific transformation by c-fms activating loop mutations is attributable to constitutive receptor degradation

Growth Hormone Receptor and Insulin-like Growth Factor-I Immunoreactivity in Osteoclast-like Cells During Tooth Eruption in the Toothless (Osteopetrotic) Rat Following Treatment with Colony-Stimulating Factor-1

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