An improved protocol for large scale production of high purity ‘Fc’ fragment of human immunoglobulin G (IgG-Fc)

Tandale, Jatin B.; Badgujar, Shamkant B.; Tandale, Babasaheb U.; Angre, Unmesh; Daftary, Siddharth B.; Lala, Sanjeev; Gaur, Vinod P.

doi:10.1016/j.jchromb.2020.122400

Cited by 2 publications

(2 citation statements)

References 46 publications

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“…Protein-G/A purified TβRII-SE/Fc allowed us to initially determine the apparent molecular mass of the monomer as a broad band of approximately 37 kDa. This observation is in agreement with the sum of the predicted molecular weight of TβRII-SE (6.5 kDa), plus the apparent molecular mass of the IgG1-Fc domain (31-32 kDa) in SDS-PAGE under reducing conditions, as a result of glycosylation (Tandale et al, 2020).…”

Section: Discussionsupporting

confidence: 88%

A Novel Splice Variant of Human TGF-β Type II Receptor Encodes a Soluble Protein and Its Fc-Tagged Version Prevents Liver Fibrosis in vivo

Bertolio

Colla

Carrea

et al. 2021

Front. Cell Dev. Biol.

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We describe, for the first time, a new splice variant of the human TGF-β type II receptor (TβRII). The new transcript lacks 149 nucleotides, resulting in a frameshift and the emergence of an early stop codon, rendering a truncated mature protein of 57 amino acids. The predicted protein, lacking the transmembrane domain and with a distinctive 13-amino-acid stretch at its C-terminus, was named TβRII-Soluble Endogenous (TβRII-SE). Binding predictions indicate that the novel 13-amino-acid stretch interacts with all three TGF-β cognate ligands and generates a more extensive protein–protein interface than TβRII. TβRII-SE and human IgG1 Fc domain were fused in frame in a lentiviral vector (Lv) for further characterization. With this vector, we transduced 293T cells and purified TβRII-SE/Fc by A/G protein chromatography from conditioned medium. Immunoblotting revealed homogeneous bands of approximately 37 kDa (reduced) and 75 kDa (non-reduced), indicating that TβRII-SE/Fc is secreted as a disulfide-linked homodimer. Moreover, high-affinity binding of TβRII-SE to the three TGF-β isoforms was confirmed by surface plasmon resonance (SPR) analysis. Also, intrahepatic delivery of Lv.TβRII-SE/Fc in a carbon tetrachloride-induced liver fibrosis model revealed amelioration of liver injury and fibrosis. Our results indicate that TβRII-SE is a novel member of the TGF-β signaling pathway with distinctive characteristics. This novel protein offers an alternative for the prevention and treatment of pathologies caused by the overproduction of TGF-β ligands.

show abstract

Section: Discussionsupporting

confidence: 88%

A Novel Splice Variant of Human TGF-β Type II Receptor Encodes a Soluble Protein and Its Fc-Tagged Version Prevents Liver Fibrosis in vivo

Bertolio

Colla

Carrea

et al. 2021

Front. Cell Dev. Biol.

View full text Add to dashboard Cite

show abstract

“…This observation is in agreement with the sum of the predicted molecular weight of TβRII-SE (6.5 kDa), plus the apparent molecular mass of IgG1-Fc domain (31-32 kDa) in SDS-PAGE under reducing conditions, as a result of glycosylation. 34…”

Section: Discussionmentioning

confidence: 99%

A novel splice variant of human TGF-β type II receptor encodes a soluble protein and its Fc-tagged version prevents liver fibrosisin vivo

Bertolio

Colla

Carrea

et al. 2021

Preprint

View full text Add to dashboard Cite

We describe, for the first time, a new splice variant of the human TGF-β type II receptor (TβRII). The new transcript lacks 149 nucleotides, causing a frameshift with the appearance of an early stop codon, rendering a truncated mature protein of 57 amino acids. The predicted protein, lacking the transmembrane domain and with a distinctive 13 amino acid stretch in the C-terminus, was named TβRII-Soluble Endogenous (TβRII-SE). Binding predictions indicated that the novel 13 amino acid stretch interacts with all three TGF-β cognate ligands and generate a more extensive protein-protein interface than TβRII. TβRII-SE and human IgG1 Fc-domain, were fused in frame in a lentiviral vector (Lv) for further characterization. With this vector, we transduced 293T cells and purified TβRII-SE/Fc by A/G protein chromatography from conditioned medium. Immunoblotting revealed homogeneous bands of approximately 37 kDa (reduced) and 75 kD (non-reduced), indicating that TβRII-SE/Fc is secreted as a disulphide-linked homodimer. Moreover, high affinity binding of TβRII-SE to the three TGF-β isoforms was confirmed by Surface Plasmon Resonance (SPR) analysis. Also, intrahepatic delivery of Lv.TβRII-SE/Fc in a carbon tetrachloride-induced liver fibrosis model revealed amelioration of liver injury and fibrosis. Our results indicate that TβRII-SE is a novel member of the TGF-β signaling pathway with distinctive characteristics. This novel protein offers an alternative tool for the prevention and treatment of pathologies caused by the overproduction of TGF-β ligands.

show abstract

An improved protocol for large scale production of high purity ‘Fc’ fragment of human immunoglobulin G (IgG-Fc)

Cited by 2 publications

References 46 publications

A Novel Splice Variant of Human TGF-β Type II Receptor Encodes a Soluble Protein and Its Fc-Tagged Version Prevents Liver Fibrosis in vivo

A Novel Splice Variant of Human TGF-β Type II Receptor Encodes a Soluble Protein and Its Fc-Tagged Version Prevents Liver Fibrosis in vivo

A novel splice variant of human TGF-β type II receptor encodes a soluble protein and its Fc-tagged version prevents liver fibrosisin vivo

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