Expression, purification and characterization of BGERII: A novel pan-TGFβ inhibitor

Erik V. Verona, Yuping Tang, Thomas K. Millstead, Andrew P. Hinck, Joseph K. Agyin, Luzhe Sun

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

Transforming growth factor beta (TGFβ) isoforms are known to be upregulated during the progression of some diseases. They have been shown to stimulate invasion and metastasis during carcinogenesis and promote many pathological fibrotic diseases when overstimulated. This involvement in late-stage carcinoma and pathological fibrosis makes TGFβ isoforms prime targets for therapeutic intervention. Although soluble ectodomains of TGFβ type II (RII) and betaglycan (BG) have been utilized as TGFβ inhibitors, their antagonistic potency against different TGFβ isoforms varies considerably because RII does not appreciably bind to TGFβ2 whereas BG binds weakly to TGFβ1 and TGFβ3. In this study, we have successfully constructed and expressed a recombinant fusion protein containing the endoglin domain of BG (BGE) and the extracellular domain of RII. The fusion protein (named BGERII) was purified from bacterial inclusion bodies by immobilized metal ion chromatography, refolded and characterized. It bound with higher affinity to TGFβ1 and TGFβ3 than a commercially available soluble RII and to TGFβ2 than a commercially available soluble BG. More significantly, whereas BGE or RII alone showed no antagonistic activity towards TGFβ2, BGERII inhibited the signaling of both TGFβ1 and TGFβ2 in cell-based assays including TGFβ-induced phosphorylation of Smad2 and Smad3, and transcription from a TGFβ-responsive promoter more effectively than equimolar concentrations of either RII or BG. After further purification by gel filtration chromatography, BGERII was found to have greater activity than other potent TGFβ inhibitors in blocking the signaling of TGFβ1 and TGFβ3. Thus, BGERII is a potent pan-TGFβ inhibitor in vitro and has potential for blocking TGFβ-induced pathogenesis in vivo.

Original languageEnglish (US)
Pages (from-to)463-473
Number of pages11
JournalProtein Engineering, Design and Selection
Volume21
Issue number7
DOIs
StatePublished - Jul 2008

Fingerprint

Transforming Growth Factor beta
Purification
Protein Isoforms
Fusion reactions
Intercellular Signaling Peptides and Proteins
Recombinant Fusion Proteins
Proteins
Ion chromatography
Phosphorylation
Inclusion Bodies
Transcription
Chromatography
Gel Chromatography
Metal ions
Disease Progression
Assays
Carcinogenesis
Fibrosis
Gels
Metals

Keywords

  • Inhibitors
  • Recombinant proteins
  • Soluble receptors
  • TGFβ

ASJC Scopus subject areas

  • Biochemistry
  • Biotechnology

Cite this

Expression, purification and characterization of BGERII : A novel pan-TGFβ inhibitor. / Verona, Erik V.; Tang, Yuping; Millstead, Thomas K.; Hinck, Andrew P.; Agyin, Joseph K.; Sun, Luzhe.

In: Protein Engineering, Design and Selection, Vol. 21, No. 7, 07.2008, p. 463-473.

Research output: Contribution to journalArticle

Verona, Erik V. ; Tang, Yuping ; Millstead, Thomas K. ; Hinck, Andrew P. ; Agyin, Joseph K. ; Sun, Luzhe. / Expression, purification and characterization of BGERII : A novel pan-TGFβ inhibitor. In: Protein Engineering, Design and Selection. 2008 ; Vol. 21, No. 7. pp. 463-473.
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