User:Cheuk Ka Tong/Sandbox2

Pore Proteins

In light of the specification of the Cell By Date system to last for at least 24 hours, the team has looked into different ways to prolong the cell-free expression system. Based on A vesicle bioreactor as a step toward an artificial cell assembly by Vincent Noireaux and Albert Libchaber, transcription-translation of plasmid genes stops after 2 hours, whille encapsulaiton of cell extract into phospholipid vesicles surrounded by feeding solution prolongs the expression system by up to 5 hours. When pore proteins are expressed inside the vesicles to create selective permeability for nutrients, the system sustains expression for up to 4 days. Having separately achieved in-vitro expression and formation of vesicles, the team is now working on putting cell extract into vesicles. This page looks at the possible candidates of pore proteins we can employ to attain the desired selective permeability in the vesicles.

Specifications for pore proteins

• Non-toxic
• Accessible
• Affordable
• Compatible with lipids used (DOPC/POPC)
• Forms a pore of ~1.4nm in diameter, or
• Molecular mass cut-off of ~3kDa

List of pore protein candidates

No.	Pore protein	Description	Toxicity	Pore size	Compatibility with lipids	Accessibility/affordability
1	α-Hemolysin toxin	Derived from S. aureus Expressed as a monomer and assembles into heptamer at the membrane Channel comprises a 14-strand beta barrel Pore remains open at neutral pH and high ionic strength	Toxic	1.4nm in diameter Molecular mass cut-off of 3kDa	Forms pores in lipid bilayers composed of phosphatidylcholine/ phosphatidylethanolamine
2	Mitochondrial porin (VDAC)	Located in the mitochondrial outer membrane Monomeric protein	Non-toxic	3nm in diameter Molecular mass cut-off of 3.4 kDa Large enough for dsDNA to pass through		Available from Abcam Product no. ab16131 GBP99.00 for 0.1mg
3	OmpF porin	Located in the E. coli outer membrane Homotrimeric protein Each monomer consists of a 16-stranded beta-barrel	Non-toxic	1.2nm in diameter Large enough for ssDNA to pass through
4	OmpG porin	Located in the E. coli outer membrane Most likely functions as a monomer Consists of 14 beta-strands	Non-toxic	1.3nm in diameter
5	OmpC porin	Located in the E. coli outer membrane	Non-toxic
6	PhoE porin	Located in the E. coli outer membrane	Non-toxic
7	Magainin 2	Derived from African clawed frog Xenopus laevis Peptide antibiotic with antibacterial and antiparasitic activities Assumes an amphiphilic helix when bound to the lipid FW = 2466.90	Non-toxic	1.6nm in diameter	Forms pores in DOPC/DOPG lipid bilayers Forms pores in POPC/DOPS or POPC/DOPG Does not form pores in POPC alone Forms pores in anionic lipids but not in zwitterionic lipids Used in 3-10μM concentrations	Available from Sigma Product no. M7402 GBP285.00 for 1mg Also available from Cambridge Bioscience Product no. ANA20640 GBP112.00 for 1mg
8	Magainin 1	Derived from African clawed frog Xenopus laevis Peptide antibiotic with antibacterial and antiparasitic activities	Non-toxic			Available from Sigma Product no. M7152 GBP290.50 for 1mg Also available from Cambridge Bioscience Product no. ANA20792 GBP112.00 for 1mg
9	Cecropin A	Peptide antibiotic with antibacterial and antiparasitic activities	Non-toxic			Available from Sigma Product no. C6830 GBP83.10 for 0.1mg