1 / 9

Proposal: pHLIP generation in E.coli

Proposal: pHLIP generation in E.coli. By: Brandon Lee and Ahad Waraich. A peptide found in Bacteriorhodopsin. pHLIP = pH Low-insertion peptide molecule Sequence: AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTCG

lilika
Télécharger la présentation

Proposal: pHLIP generation in E.coli

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  1. Proposal: pHLIP generation in E.coli By: Brandon Lee and AhadWaraich

  2. A peptide found in Bacteriorhodopsin. • pHLIP = pH Low-insertion peptide molecule • Sequence: AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTCG • - At low pH environments, spontaneous insertion (into membrane bilayers) and formation of transbilayer alpha-helices occurs. What is pHLIP?

  3. A Bit of History and Why we should care • pHLIP first discovered by the Donald M. Engelman group at Yale University and collaborating members from the University of Rhode Island. • High extracellular acidity is associated with many pathological conditions: drug delivery and cell imaging can be done via pHLIP! High acidity is not observed in normal cellular environments • - tumors, • - infarcts, • - stroke-afflicted tissue, • - atherosclerotic lesions

  4. How it works: What happens • Forms transmembrane alpha-helix: becomes rigid at low pH like nano-syringe • Inserts in low pH environments but only associates at pH > 7.0; Triggered by the increase of the peptide hydrophobicity due to the protonation of Asp residues induced by low pH. • N terminus stays outside, C terminus of the peptide is translocated across the bilayer • - Previous experiments show that disulfide bonding to the c-terminus can be cleaved by reducing environment within cell. Can attach dye’s or other molecules to this end.

  5. Results of Several Studies Done • The delivery of phalloidin into cells by pHLIP. (a) Fluorescence images of HeLa cells incubated (for 1 h) with a pHLIP–S–S–Ph–TRITC cleavable construct • (2 M) at pH 7.4 • - Strong fluorescence of actin filaments was observed after pH 6.5 • incubation. Tumor and inflammation imaging: - (b) Overlay of pHLIP-Cy5.5 fluorescence and light images of mice bearing a tumor (7mmin diameter, 12 d after 106 cell implant)

  6. What remains to be known? • The size of molecules that can be attached to pHLIP remains to be known • Polarity of molecules that can be attached also remains to be studied • What available drugs and imaging techniques can we apply to this molecule? • Overall, this molecule has not been explored in too much detail

  7. What can we do? • Recent experiments by the Engelman group have shown that near-infrared fluorescence imaging works in mouse cancers and rat inflammatory arthiritis models using pHLIP as a delivery device • NIR fluorescent dyes can be conjugated with Cys or Lys residues placed on pHLIP N-terminus (outside of cell upon insertion). • - Dyes that can be used • - Cy5.5 or Alexa750 • Possibly initiatives: • Since NIR range (700 – 900 nm) can propagate through tissues in organs and whole bodies, why not use this for early tumor detection? • Another direction: Is there some way to make this delivery device easily accessible? Produced in large quantities?

  8. Why Synthetic Biology? • pHLIP related to a peptide in bacteriorhodopsin found in halobacteria (not a model organism) • pHLIP has been created through solid-phase peptide synthesis (SPPS) via 9-fluorenylmethyloxycarbonyl (FMOC) chemistry, purified via reverse-phase chromatography • FMOC solid-phase peptide synthesis: Treat small, solid beads with linkers (functional units) that can be used to make peptide chains

  9. Why Synthetic Biology? • SPPS may be “limited by yield” • Perhaps synthetic biology (generate it in E. coli, etc.) could be better • Even if there are no apparent problems with SPPS, there is no harm in trying to create the protein in a new way (like synthetic biology, with E. coli); something advantageous about a synthetic biology method could arise

More Related