A chromatographically purified human TGF-β1 fraction from virally inactivated platelet lysates

T. Burnouf, C. W. Chang, Y. P. Kuo, Y. W. Wu, Y. H. Tseng, C. Y. Su

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

Background and Objectives TGF-β1 exerts important physiological functions in osteogenesis and chondrogenesis and may be of therapeutic interest. The aim of this work was to develop a scalable purification process of TGF-β1 from virally inactivated human platelets. Study Design and Methods Apheresis platelet concentrates (N=12) were solvent/detergent (S/D) treated (1% TnBP/1% Triton X-45; 31°C) and the resulting platelet lysates were clarified by oil extraction and centrifugation, then chromatographed on an anion-exchange DEAE-Sepharose Fast-Flow column equilibrated in a PBS buffer, pH 7·5. The column was washed to eliminate unbound proteins and the S/D agents. Bound proteins were eluted using a 1M NaCl-PBS buffer pH 7·5 (DEAE-eluate). The content in TGF-β1, PDGF-AB, VEGF, IGF-1, EGF, and b-FGF was measured by ELISA. Proteins, lipids, and S/D agents were assessed. Protein profile was determined by SDS-PAGE under reduced or non-reduced conditions. Results Most proteins, including albumin and immunoglobulins G, A, and M did not bind to the DEAE column as evidenced also by SDS-PAGE. Essentially all PDGF, VEGF, and IGF were in the breakthrough. The DEAE-eluate contained close to 60% of the TGF-β1 at a mean concentration of about 102ng/ml, whereas EGF, b-FGF were at about 0·72 and 0·18ng/ml, respectively. The content in TnBP and Triton X-45 was

Original languageEnglish
Pages (from-to)215-220
Number of pages6
JournalVox Sanguinis
Volume101
Issue number3
DOIs
Publication statusPublished - Oct 2011

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Blood Platelets
Detergents
Octoxynol
Proteins
Epidermal Growth Factor
Vascular Endothelial Growth Factor A
Polyacrylamide Gel Electrophoresis
Buffers
Chondrogenesis
Blood Component Removal
Insulin-Like Growth Factor I
Centrifugation
Osteogenesis
Sepharose
Anions
Immunoglobulin M
Albumins
Oils
Immunoglobulin G
Enzyme-Linked Immunosorbent Assay

Keywords

  • Chondrogenesis
  • Fractionation
  • Osteogenesis
  • Platelet
  • Solvent-detergent
  • TGF-β1

ASJC Scopus subject areas

  • Hematology

Cite this

A chromatographically purified human TGF-β1 fraction from virally inactivated platelet lysates. / Burnouf, T.; Chang, C. W.; Kuo, Y. P.; Wu, Y. W.; Tseng, Y. H.; Su, C. Y.

In: Vox Sanguinis, Vol. 101, No. 3, 10.2011, p. 215-220.

Research output: Contribution to journalArticle

Burnouf, T. ; Chang, C. W. ; Kuo, Y. P. ; Wu, Y. W. ; Tseng, Y. H. ; Su, C. Y. / A chromatographically purified human TGF-β1 fraction from virally inactivated platelet lysates. In: Vox Sanguinis. 2011 ; Vol. 101, No. 3. pp. 215-220.
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abstract = "Background and Objectives TGF-β1 exerts important physiological functions in osteogenesis and chondrogenesis and may be of therapeutic interest. The aim of this work was to develop a scalable purification process of TGF-β1 from virally inactivated human platelets. Study Design and Methods Apheresis platelet concentrates (N=12) were solvent/detergent (S/D) treated (1{\%} TnBP/1{\%} Triton X-45; 31°C) and the resulting platelet lysates were clarified by oil extraction and centrifugation, then chromatographed on an anion-exchange DEAE-Sepharose Fast-Flow column equilibrated in a PBS buffer, pH 7·5. The column was washed to eliminate unbound proteins and the S/D agents. Bound proteins were eluted using a 1M NaCl-PBS buffer pH 7·5 (DEAE-eluate). The content in TGF-β1, PDGF-AB, VEGF, IGF-1, EGF, and b-FGF was measured by ELISA. Proteins, lipids, and S/D agents were assessed. Protein profile was determined by SDS-PAGE under reduced or non-reduced conditions. Results Most proteins, including albumin and immunoglobulins G, A, and M did not bind to the DEAE column as evidenced also by SDS-PAGE. Essentially all PDGF, VEGF, and IGF were in the breakthrough. The DEAE-eluate contained close to 60{\%} of the TGF-β1 at a mean concentration of about 102ng/ml, whereas EGF, b-FGF were at about 0·72 and 0·18ng/ml, respectively. The content in TnBP and Triton X-45 was",
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N2 - Background and Objectives TGF-β1 exerts important physiological functions in osteogenesis and chondrogenesis and may be of therapeutic interest. The aim of this work was to develop a scalable purification process of TGF-β1 from virally inactivated human platelets. Study Design and Methods Apheresis platelet concentrates (N=12) were solvent/detergent (S/D) treated (1% TnBP/1% Triton X-45; 31°C) and the resulting platelet lysates were clarified by oil extraction and centrifugation, then chromatographed on an anion-exchange DEAE-Sepharose Fast-Flow column equilibrated in a PBS buffer, pH 7·5. The column was washed to eliminate unbound proteins and the S/D agents. Bound proteins were eluted using a 1M NaCl-PBS buffer pH 7·5 (DEAE-eluate). The content in TGF-β1, PDGF-AB, VEGF, IGF-1, EGF, and b-FGF was measured by ELISA. Proteins, lipids, and S/D agents were assessed. Protein profile was determined by SDS-PAGE under reduced or non-reduced conditions. Results Most proteins, including albumin and immunoglobulins G, A, and M did not bind to the DEAE column as evidenced also by SDS-PAGE. Essentially all PDGF, VEGF, and IGF were in the breakthrough. The DEAE-eluate contained close to 60% of the TGF-β1 at a mean concentration of about 102ng/ml, whereas EGF, b-FGF were at about 0·72 and 0·18ng/ml, respectively. The content in TnBP and Triton X-45 was

AB - Background and Objectives TGF-β1 exerts important physiological functions in osteogenesis and chondrogenesis and may be of therapeutic interest. The aim of this work was to develop a scalable purification process of TGF-β1 from virally inactivated human platelets. Study Design and Methods Apheresis platelet concentrates (N=12) were solvent/detergent (S/D) treated (1% TnBP/1% Triton X-45; 31°C) and the resulting platelet lysates were clarified by oil extraction and centrifugation, then chromatographed on an anion-exchange DEAE-Sepharose Fast-Flow column equilibrated in a PBS buffer, pH 7·5. The column was washed to eliminate unbound proteins and the S/D agents. Bound proteins were eluted using a 1M NaCl-PBS buffer pH 7·5 (DEAE-eluate). The content in TGF-β1, PDGF-AB, VEGF, IGF-1, EGF, and b-FGF was measured by ELISA. Proteins, lipids, and S/D agents were assessed. Protein profile was determined by SDS-PAGE under reduced or non-reduced conditions. Results Most proteins, including albumin and immunoglobulins G, A, and M did not bind to the DEAE column as evidenced also by SDS-PAGE. Essentially all PDGF, VEGF, and IGF were in the breakthrough. The DEAE-eluate contained close to 60% of the TGF-β1 at a mean concentration of about 102ng/ml, whereas EGF, b-FGF were at about 0·72 and 0·18ng/ml, respectively. The content in TnBP and Triton X-45 was

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