N‐terminal‐methionylated interleukin‐1β has reduced receptor‐binding affinity

Wingfield, Paul T.; Graber, Pierre; Gronenborn, Angela M.; MacDonald, H. Robson

doi:10.1016/0014-5793(87)80133-3

Cited by 24 publications

(12 citation statements)

References 16 publications

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“…The retention of the initiating Met does not impair bioactivity in the case of recombinant cytokines such as granulocyte-macrophage colony-stimulating factor (25), interleukin-2 (26), and interleukin-5 (27). However some 5-10-fold shifts in receptor binding affinity have been seen in certain cases such as hirudin (28) and interleukin-1␤ (29).…”

Section: Discussionmentioning

confidence: 99%

Extension of Recombinant Human RANTES by the Retention of the Initiating Methionine Produces a Potent Antagonist

Proudfoot¹,

Power²,

Hoogewerf³

et al. 1996

Journal of Biological Chemistry

397

288

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Extension of recombinant human RANTES by a single residue at the amino terminus is sufficient to produce a potent and selective antagonist. RANTES is a proinflammatory cytokine that promotes cell accumulation and activation in chronic inflammatory diseases. When mature RANTES was expressed heterologously in Escherichia coli, the amino-terminal initiating methionine was not removed by the endogenous amino peptidases. This methionylated protein was fully folded but completely inactive in RANTES bioassays of calcium mobilization and chemotaxis of the promonocytic cell line THP-1. However, when assayed as an antagonist of both RANTES and macrophage inflammatory polypeptide-1␣ (MIP-1␣) in these assays, the methionylated RANTES (Met-RANTES) inhibited the actions of both chemokines. T cell chemotaxis was similarly inhibited. The antagonistic effect was selective since Met-RANTES had no effect on interleukin-8-or monocyte chemotractant protein-1-induced responses in these cells. Met-RANTES can compete with both [ RANTES is a member of a large family of cytokines, known as chemokines, which have the ability to recruit and activate a wide variety of proinflammatory cell types (1). They are small polypeptides of 8 -10 kDa and have been further classified into CXC or CC chemokines based on the spacings of the cysteine residues proximal to the amino terminus. CXC chemokines primarily activate neutrophils, whereas CC chemokines have effects on several leucocyte cell types. RANTES is a CC chemokine, and in vitro it can produce chemotaxis and activation of monocytes, eosinophils, and T cells, particularly CD4 ϩ CD45RO ϩ (memory) T cells (2), but not neutrophils. These results imply a role for RANTES in diseases such as allergen induced late phase skin reactions or in allergic asthma. This hypothesis is strengthened by the fact that large amounts of RANTES are found in nasal polyp tissues, which are rich in infiltrating eosinophils (3). In addition, injection of RANTES into dog skin has been shown to induce a large eosinophilic infiltrate in vivo (4), and migration of human T lymphocytes was observed on injection of human RANTES into a human/severe combined immune deficiency mouse model (5).MIP-1␣ shares an overlapping cell-type specificity with RANTES in vitro (6, 7) and has been shown to elicit an inflammatory response mediated through mast cell degranulation in vivo (8). A common receptor for these two CC chemokines has been cloned (9, 10) and is a member of the seven transmembrane G-protein linked receptor family. Recombinant expression of the receptor has shown that it can transduce a functional response on stimulation by both chemokines.We report the purification of human RANTES expressed heterologously in Escherichia coli. In this system, the protein retains its initiating methionine residue, which renders it inactive as an agonist, while enabling it to antagonize effects induced both by RANTES and MIP-1␣. It is able to compete for binding of both the radiolabeled ligands on THP-1 cells and to the recombinant RANTES/MIP-1...

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Section: Discussionmentioning

confidence: 99%

Extension of Recombinant Human RANTES by the Retention of the Initiating Methionine Produces a Potent Antagonist

Proudfoot¹,

Power²,

Hoogewerf³

et al. 1996

Journal of Biological Chemistry

397

288

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“…Previous work comparing N-terminal-methionylated and nonmethionylated wild-type IL-1b showed significant differences in the isoelectric point and receptor binding activity between the two versions of this cytokine. 69 It is possible that the activity and/or cellular uptake of IL-3 were also strongly affected by N-terminal methionylation and led to significant differences in Mk expansion.…”

Section: Discussionmentioning

confidence: 99%

Three-StageEx VivoExpansion of High-Ploidy Megakaryocytic Cells: Toward Large-Scale Platelet Production

Panuganti

Schlinker

Lindholm

et al. 2013

Tissue Engineering Part A

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“…E. coli expresses endogenous aminopeptidases, including aminopeptidases M and P, which can be responsible for proteolytic degradation or post‐translational processing [8, 9]. Although some of these aminopeptidases are necessary for cell maintenance [10] or product activity [11, 12], they can also lead to undesirable product variants and reduced quality [13]. Based on the target product profile of EBI‐005, it was essential to control host aminopeptidase activity during fermentation and reduce product variant levels.…”

Section: Introductionmentioning

confidence: 99%

Reduction of product‐related species during the fermentation and purification of a recombinant IL‐1 receptor antagonist at the laboratory and pilot scale

Schirmer¹,

Golden²,

et al. 2013

Biotechnology Journal

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Through a parallel approach of tracking product quality through fermentation and purification development, a robust process was designed to reduce the levels of product-related species. Three biochemically similar product-related species were identified as byproducts of host-cell enzymatic activity. To modulate intracellular proteolytic activity, key fermentation parameters (temperature, pH, trace metals, EDTA levels, and carbon source) were evaluated through bioreactor optimization, while balancing negative effects on growth, productivity, and oxygen demand. The purification process was based on three non-affinity steps and resolved product-related species by exploiting small charge differences. Using statistical design of experiments for elution conditions, a high-resolution cation exchange capture column was optimized for resolution and recovery. Further reduction of product-related species was achieved by evaluating a matrix of conditions for a ceramic hydroxyapatite column. The optimized fermentation process was transferred from the 2-L laboratory scale to the 100-L pilot scale and the purification process was scaled accordingly to process the fermentation harvest. The laboratory- and pilot-scale processes resulted in similar process recoveries of 60 and 65%, respectively, and in a product that was of equal quality and purity to that of small-scale development preparations. The parallel approach for up- and downstream development was paramount in achieving a robust and scalable clinical process.

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N‐terminal‐methionylated interleukin‐1β has reduced receptor‐binding affinity

Cited by 24 publications

References 16 publications

Extension of Recombinant Human RANTES by the Retention of the Initiating Methionine Produces a Potent Antagonist

Extension of Recombinant Human RANTES by the Retention of the Initiating Methionine Produces a Potent Antagonist

Three-StageEx VivoExpansion of High-Ploidy Megakaryocytic Cells: Toward Large-Scale Platelet Production

Reduction of product‐related species during the fermentation and purification of a recombinant IL‐1 receptor antagonist at the laboratory and pilot scale

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