Proposal: pHLIP generation in E.coli

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Proposal pHLIP generation in E.coli

• By Brandon Lee and Ahad Waraich

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What is pHLIP?

• 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.

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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

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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 gt 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
  dyes or other molecules to this end.

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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 pHLIPSSPhTRITC 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)
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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

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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?

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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-fluorenylmethyloxyc
  arbonyl (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

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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