Detergent-solubilized Patched purified from Sf9 cells fails to interact strongly with cognate Hedgehog or Ihog homologs

Abstract

Patched (Ptc) is a twelve-pass transmembrane protein that functions as a receptor for the Hedgehog (Hh) family of morphogens. In addition to Ptc, several accessory proteins including the CDO/Ihog family of co-receptors are necessary for proper Hh signaling. Structures of Hh proteins bound to members of the CDO/Ihog family are known, but the nature of the full Hh receptor complex is not well understood. We have expressed the Drosophila Patched and Mouse Patched-1 proteins in Sf9 cells and find that Sonic Hedgehog will bind to Mouse Patched-1 in isolated Sf9 cell membranes but that purified, detergent-solubilized Ptc proteins do not interact strongly with cognate Hh and CDO/Ihog homologs. These results may reflect a nonnative conformation of detergent-solubilized Ptc or that an additional factor or factors lost during purification are required for high-affinity Ptc binding to Hh.

Structural Basis for Cholesterol Transport-like Activity of the Hedgehog Receptor Patched

“…Proteins were expressed in E. coli strain BL21(DE3) and purified according to the protocol described in ref. (Cleveland et al, 2014a), with minor modifications. In brief, for the cysteine variant, 2 mM TCEP was included in all the buffers used during purification, to keep the cysteine reduced.…”

“…The full-length mouse PTCH1 protein is poorly expressed and biochemically unstable (Cleveland et al, 2014b). We sought to identify a more stable protein variant by using FSEC ( f luorescence detection s ize e xclusion c hromatography (Kawate and Gouaux, 2006)) to screen constructs from which potential destabilizing sequences were deleted.…”

A cell-free antigen processing system informs HIV-1 epitope selection and vaccine design

“…Sf9 and Hi5 cells were used to produce recombinant DR1 and DM. Cells were grown as previously described ( Kim et al, 2014 ; Narayan et al, 2007 ) in ISFM media ( Cleveland et al, 2014 ; Inlow et al, 1989 ) at a density of 0.3–0.5 million cells/ml at 27°C, with regular passaging every 3–4 d.…”

“…Baculovirus DNA (BaculoGold; PharMingen) and transfer vectors carrying DR α- and β-chains were transfected together into Sf9 insect cells to produce recombinant viruses. Recombinant viruses were passaged three times before being used to infect High Five cells in ISFM media ( Cleveland et al, 2014 ; Inlow et al, 1989 ). DR1 proteins were purified from culture supernatants using immunoaffinity chromatography with a monoclonal antibody L243 to DR1 (purified from HB-55 hybridoma; American Type Culture Collection).…”

Design and characterization of a photo-activatable hedgehog probe that mimics the natural lipidated form

“…Demonstrating direct binding of Hh to the HSPG glypican-3 was not possible with purified components [43] but has been demonstrated for Shh binding to heparin and chondroitin sulfate [38]. Interestingly, recent attempts to recapitulate Shh binding to detergent-solubilized Ptc has proved difficult suggesting that additional factors may be involved for high affinity binding of Hh to Ptc [44]. …”