September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Expression, purification and characterization of a membrane-bound receptor for PEDF
Author Affiliations & Notes
  • Jeanee Bullock
    National Eye Institute, Bethesda, Maryland, United States
    Biochemistry and Molecular & Cellular Biology, Georgetown University, Washington, District of Columbia, United States
  • Preeti Subramanian
    National Eye Institute, Bethesda, Maryland, United States
  • S Patricia Becerra
    National Eye Institute, Bethesda, Maryland, United States
  • Footnotes
    Commercial Relationships   Jeanee Bullock, None; Preeti Subramanian, None; S Patricia Becerra, None
  • Footnotes
    Support  Intramural Research Program of the NEI-NIH Project # ZIA EY000306
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 4806. doi:
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    • Get Citation

      Jeanee Bullock, Preeti Subramanian, S Patricia Becerra; Expression, purification and characterization of a membrane-bound receptor for PEDF. Invest. Ophthalmol. Vis. Sci. 2016;57(12):4806.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Pigment epithelium-derived factor-receptor (PEDF-R) acts in retina survival. It is a cell-surface protein receptor with phospholipase activity that is stimulated by the neuroprotective factor PEDF. Overexpression of PEDF-R yields low levels in mammalian expression systems. The present study tests the hypothesis that PEDF-R overexpressed in E.coli will retain the ability to bind PEDF and remain enzymatically active.

Methods : The human PNPLA2 gene was used for the preparation of recombinant PEDF-R polypeptides. Expression vectors for full-length and a C-terminal truncated PEDF-R were engineered using p-EXP1-DEST plasmid and used to transform BL21 (DE3) E. coli cells to make them resistant to ampicillin. Isopropyl β-D-1-thiogalactopyranoside was used to induce heterologous gene expression at various temperatures. Purification of the recombinant proteins from cell extracts included sonication, differential centrifugation, solubilization with chaotropic agents and detergents, and ion exchange chromatography. SDS-PAGE, western blots, PEDF binding assays and lipase activity assays were performed.

Results : Bacterially-derived recombinant PEDF-R polypeptide versions were obtained in large amounts in inclusion bodies. The overexpression efficacy yielded levels of recombinant PEDF-R polypeptides at about 7 mg/g wet cell pellet. Recombinant full-length and truncated PEDF-R migrated by SDS-PAGE as expected from their derived length and immunoreacted with specific antibodies to PEDF-R and a tag Anti-Xpress. Urea solutions at 2M, 4M, 6M and 8 M urea were used to solubilize the polypeptides. PEDF-R polypeptides became soluble only in 6M and 8M urea. Highly purified proteins were obtained after cation- and anion-exchange column chromatography. The PEDF-R polypeptides were also soluble after dialysis against buffers containing 0.5% Tween20, 0.1% Triton X-100, and 3 mM deoxycholate. Fractions containing recombinant PEDF-R exhibited phospholipase activity. Refolded PEDF-R protein bound to PEDF by pull-down assays.

Conclusions : Bacterial overexpression yielded high amounts of recombinant PEDF-R polypeptides that surpassed those described from the mammalian systems. The PEDF-R polypeptides were refolded retaining biochemical activity like the mammalian protein.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

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