High-Level Expression of Recombinant Fc Chimeric Proteins in Suspension Cultures of Stably Transfected J558L Cells

Howard, Mark; Lodge, Anthony P.; Reed, James E.; McNamee, Christine J.; Moss, Darren M.

doi:10.2144/02326st03

Cited by 5 publications

(4 citation statements)

References 14 publications

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“…1). Because J558L murine plasmacytoma cells are capable of producing greater amounts of ectopic fusion protein compared with CHO or HEK293 cells (27), J558L cells were transfected, drug-selected, subcloned, and assayed for Gal-1hFc expression by RT-PCR and Western blotting. Stable clones secreted a Gal-1hFc and mutant protein, which was isolated by protein G affinity chromatography and resolved at the predicted sizes of 40.7 and 81.4 kDa, under reducing and nonreducing conditions, respectively (Fig.…”

Section: Resultsmentioning

confidence: 99%

Development of a Nascent Galectin-1 Chimeric Molecule for Studying the Role of Leukocyte Galectin-1 Ligands and Immune Disease Modulation

Cedeno-Laurent

Barthel

Opperman³

et al. 2010

The Journal of Immunology

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Galectin-1 (Gal-1), a β-galactoside–binding lectin, plays a profound role in modulating adaptive immune responses by altering the phenotype and fate of T cells. Experimental data showing recombinant Gal-1 (rGal-1) efficacy on T cell viability and cytokine production, nevertheless, is controversial due to the necessity of using stabilizing chemicals to help retain Gal-1 structure and function. To address this drawback, we developed a mouse Gal-1 human Ig chimera (Gal-1hFc) that did not need chemical stabilization for Gal-1 ligand recognition, apoptosis induction, and cytokine modulation in a variety of leukocyte models. At high concentrations, Gal-1hFc induced apoptosis in Gal-1 ligand+ Th1 and Th17 cells, leukemic cells, and granulocytes from synovial fluids of patients with rheumatoid arthritis. Importantly, at low, more physiologic concentrations, Gal-1hFc retained its homodimeric form without losing functionality. Not only did Gal-1hFc–binding trigger IL-10 and Th2 cytokine expression in activated T cells, but members of the CD28 family and several other immunomodulatory molecules were upregulated. In a mouse model of contact hypersensitivity, we found that a non-Fc receptor-binding isoform of Gal-1hFc, Gal-1hFc2, alleviated T cell-dependent inflammation by increasing IL-4+, IL-10+, TGF-β+, and CD25high/FoxP3+ T cells, and by decreasing IFN-γ+ and IL-17+ T cells. Moreover, in human skin-resident T cell cultures, Gal-1hFc diminished IL-17+ T cells and increased IL-4+ and IL-10+ T cells. Gal-1hFc will not only be a useful new tool for investigating the role of Gal-1 ligands in leukocyte death and cytokine stimulation, but for studying how Gal-1–Gal-1 ligand binding shapes the intensity of immune responses.

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Section: Resultsmentioning

confidence: 99%

Development of a Nascent Galectin-1 Chimeric Molecule for Studying the Role of Leukocyte Galectin-1 Ligands and Immune Disease Modulation

Cedeno-Laurent

Barthel

Opperman³

et al. 2010

The Journal of Immunology

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“…Eukaryotic expression vectors were constructed that encoded a prepro-trypsin signal peptide followed by a FLAG epitope, the MUC1/Y (or MUC1/X) extracellular domains, and C-terminating with the Fc portion of human immunoglobulin (49,50). Transfection of these plasmids into cells led to secretion of fusion proteins comprising MUC1/X and MUC1/Y extracellular domains (Fig.…”

Section: Proteolytic Cleavage Occurs On Muc1 Isoform Muc1/x and Not Omentioning

confidence: 99%

The MUC1 SEA Module Is a Self-cleaving Domain

Levitin

Stern

Weiss

et al. 2005

Journal of Biological Chemistry

179

180

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MUC1, a glycoprotein overexpressed by a variety of human adenocarcinomas, is a type I transmembrane protein (MUC1/TM) that soon after its synthesis undergoes proteolytic cleavage in its extracellular domain. This cleavage generates two subunits, ␣ and ␤, that specifically recognize each other and bind together in a strong noncovalent interaction. Proteolysis occurs within the SEA module, a 120-amino acid domain that is highly conserved in a number of heavily glycosylated mucin-like proteins. Post-translational cleavage of the SEA module occurs at a site similar to that in MUC1 in the glycoproteins IgHepta and MUC3. However, as in the case of other proteins containing the cleaved SEA module, the mechanism of MUC1 proteolysis has not been elucidated. Alternative splicing generates two transmembrane MUC1 isoforms, designated MUC1/Y and MUC1/X. We demonstrated here that MUC1/X, whose extracellular domain is comprised solely of the SEA module in addition to 30 MUC1 N-terminal amino acids, undergoes proteolytic cleavage at the same site as the MUC1/TM protein. In contrast, the MUC1/Y isoform, composed of an N-terminally truncated SEA module, is not cleaved. Cysteine or threonine mutations of the MUC1/X serine residue (Ser-63) immediately C-terminal to the cleavage site generated cleaved proteins, whereas mutation of the Ser-63 residue of MUC1/X to any other of 17 amino acids did not result in cleavage. In vitro incubation of highly purified precursor MUC1/X protein resulted in self-cleavage. Furthermore, addition of hydroxylamine, a strong nucleophile, markedly enhanced cleavage. Both these features are signature characteristics of self-cleaving proteins, and we concluded that MUC1 undergoes autoproteolysis mediated by an N 3 O-acyl rearrangement at the cleavage site followed by hydrolytic resolution of the unstable ester and concomitant cleavage. It is likely that all cleaved SEA module-containing proteins follow a similar route.The MUC1 gene is highly expressed in a number of human epithelial malignancies, including breast, prostate, and colon carcinomas, as well as on the malignant plasma cells of multiple myeloma (1-9). As a well characterized tumor-associated protein, it has generated considerable interest as a tumor marker for disease prognosis (10 -14) as well as a target for tumor cell killing (15-18). Although alternative splicing can generate multiple MUC1 protein forms (19 -23), the most intensively studied MUC1 protein is a type I transmembrane protein comprised of a heavily glycosylated extracellular domain containing a tandem-repeat array, a transmembrane domain, and a cytoplasmic domain (Fig. 1, MUC1/TM) (24 -26). MUC1/TM is proteolytically cleaved soon after its synthesis, generating two subunits, ␣ and ␤, that specifically recognize each other and bind together by a strong noncovalent interaction (27).Cleavage occurs within the SEA module (28 -30), a highly conserved protein module so-called from its initial identification in a sperm protein, in enterokinase, and in agrin (31), that is found in a numb...

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“…Indeed, these cells when stably transfected, are capable of producing up to 2.7 g/L of recombinant protein (Zhou et al, 1997). Other groups achieved as much as 1-2 mg/L (Ridder et al, 1995) or 400 mg/L (Howard et al, 2002) using large-scale transient transfection of COS cells. Third, the secretory apparatus of mammalian cells has been well-studied, and this understanding aids the design of expression vectors and the secretion of intact protein molecules into cell culture medium.…”

Section: Discussionmentioning

confidence: 99%

Mammalian expression of functional autologous red cell agglutination reagents for use in diagnostic assays

Hobson‐Peters¹,

Shan²,

Hall

et al. 2010

Journal of Virological Methods

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The autologous red cell agglutination assay reagent consists of an antibody or antibody fragment of a human erythrocyte-specific monoclonal antibody (mAb) conjugated to an antigen of interest. This bi-functional reagent causes the agglutination of the patient's erythrocytes in the presence of the antigen-specific antibodies in the patient's serum. Previously, such reagents have been produced either by chemical conjugation or recombinant expression in bacteria. These protocols required laborious processes for purification and refolding. The aim of the work reported in this article was to explore the production of the agglutination assay reagent as both a single chain Fv (scFv) antibody fragment and recombinant full-length mAb, expressed in a secreted form in commonly used mammalian cell lines. The DNA encoding the anti-erythrocyte antibodies was linked to that of a diagnostic peptide from West Nile virus, which requires glycosylation for recognition by antibodies present in the sera of infected horses. The expression vectors were designed to allow the rapid directional insertion of DNA encoding other immunogenic peptides to mediate the secretion of agglutinating scFv and full-length mAb reagents from transfected mammalian cells. Stable cell lines were produced for the expression of most, but not all of the constructs. The recombinant reagents could be used directly from the cell culture media after a simple concentration step. The results indicate that further modifications to increase the yield of recombinant protein will enable the direct use of culture supernatant in diagnostic assays without further processing.

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High-Level Expression of Recombinant Fc Chimeric Proteins in Suspension Cultures of Stably Transfected J558L Cells

Cited by 5 publications

References 14 publications

Development of a Nascent Galectin-1 Chimeric Molecule for Studying the Role of Leukocyte Galectin-1 Ligands and Immune Disease Modulation

Development of a Nascent Galectin-1 Chimeric Molecule for Studying the Role of Leukocyte Galectin-1 Ligands and Immune Disease Modulation

The MUC1 SEA Module Is a Self-cleaving Domain

Mammalian expression of functional autologous red cell agglutination reagents for use in diagnostic assays

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