Proposal: pHLIP generation in E.coli

By: Brandon Lee and Ahad Waraich

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.

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

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.

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 6.5incubation.

Tumor and inflammation imaging:- (b) Overlay of pHLIP-Cy5.5 fluorescenceand light images of mice bearing a tumor (7mmin diameter, 12 d after106 cell implant)

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

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?

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

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