2 Materials
2.1 LEPS
Components
1. Lipids for assembly of LEPS include:
(a) 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC).
(b) 1,2-Dioleoyl-sn-glycero-3-phospho-(1
0 -rac-glycerol)
(DOPG).
(c) 1,2-Dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] (nickel salt) (DGS-NTANi).
(d) Cholesterol.
(e) 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N[amino(polyethylene glycol)-2000] (DSPE-PEG2000).
2. Pneumococcal Polysaccharide: Isolated 19F serotype.
3. GFP is obtained through production in recombinant E. coli
and purified chromatographically using Ni-NTA resin. The
protocol for doing so is outlined within the method
presented here.
2.2 Components
Used for Assembly
and Purification
of LEPS
1. Fisher Scientific CL-18 Sonicator.
2. Buchi Rotavapor (R-200) rotary evaporator.
3. Buchi Heating Bath (B-490).
4. Handheld Extruder with 200 nm polycarbonate filter
membrane.
5. Centrifugal Tubes with 300 kDa filters.
2.3 Protein
Production
and Purification
Materials
1. BL21(DE3) E. coli containing pET21b with GFP inserted into
the multiple cloning site is use to enable production of GFP.
2. Lysogeny broth (LB) medium: 10 g Bacto tryptone, 5 g yeast
extract, and 10 g/L NaCl.
3. LB agar plates: 10 g/L Bacto tryptone, 5 g/L yeast extract,
10 g /L NaCl, and 15 g/L agar. When bacteria are transformed
with the plasmids outlined here, resulting cultures are plated on
solid agar supplemented with antibiotics and incubated overnight at 37
C.
4. Bacterial growth medium is supplemented with ampicillin at
100 mg/L.
5. IPTG: isopropyl β-D-1-thiogalactopyranoside (100 μM) is
added to bacterial media to induce gene expression.
6. HisTrap HP column, GE Healthcare.
7. Buffer A: 50 mM Na 2 HPO 4 , 500 mM NaCl, and 10% glycerol.
8. Buffer B: 50 mM Na 2 HPO 4 , 500 mM NaCl, 10% glycerol, and
250 mM imidazole.
Liposomal Dual Antigen Delivery
479
2.1 LEPS
Components
1. Lipids for assembly of LEPS include:
(a) 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC).
(b) 1,2-Dioleoyl-sn-glycero-3-phospho-(1
0 -rac-glycerol)
(DOPG).
(c) 1,2-Dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] (nickel salt) (DGS-NTANi).
(d) Cholesterol.
(e) 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N[amino(polyethylene glycol)-2000] (DSPE-PEG2000).
2. Pneumococcal Polysaccharide: Isolated 19F serotype.
3. GFP is obtained through production in recombinant E. coli
and purified chromatographically using Ni-NTA resin. The
protocol for doing so is outlined within the method
presented here.
2.2 Components
Used for Assembly
and Purification
of LEPS
1. Fisher Scientific CL-18 Sonicator.
2. Buchi Rotavapor (R-200) rotary evaporator.
3. Buchi Heating Bath (B-490).
4. Handheld Extruder with 200 nm polycarbonate filter
membrane.
5. Centrifugal Tubes with 300 kDa filters.
2.3 Protein
Production
and Purification
Materials
1. BL21(DE3) E. coli containing pET21b with GFP inserted into
the multiple cloning site is use to enable production of GFP.
2. Lysogeny broth (LB) medium: 10 g Bacto tryptone, 5 g yeast
extract, and 10 g/L NaCl.
3. LB agar plates: 10 g/L Bacto tryptone, 5 g/L yeast extract,
10 g /L NaCl, and 15 g/L agar. When bacteria are transformed
with the plasmids outlined here, resulting cultures are plated on
solid agar supplemented with antibiotics and incubated overnight at 37
C.
4. Bacterial growth medium is supplemented with ampicillin at
100 mg/L.
5. IPTG: isopropyl β-D-1-thiogalactopyranoside (100 μM) is
added to bacterial media to induce gene expression.
6. HisTrap HP column, GE Healthcare.
7. Buffer A: 50 mM Na 2 HPO 4 , 500 mM NaCl, and 10% glycerol.
8. Buffer B: 50 mM Na 2 HPO 4 , 500 mM NaCl, 10% glycerol, and
250 mM imidazole.
Liposomal Dual Antigen Delivery
479