Recruitment of Coat Proteins to Liposomes and Peptidoliposomes - Membrane Trafficking - page 95

1. Escherichia coli BL21(DE3) (Stratagene) containing the plasmid
pET24d-Amph1 or pET24d-Amph2. The plasmids correspond
to the coding sequence of human amphiphysin 1 or 2, respec-
tively, cloned into the bacterial expression vector pET24d
(Novagen), thus C-terminally fused to a His 6 tag. Plasmids
containing mutant cDNAs are used in the same manner.
2. Luria broth (LB) with kanamycin (LB/Kana): 10 g/L bacto-
tryptone (Applichem), 5 g/L yeast extract (Applichem),
10 g/L NaCl supplemented with 50

2.4 Expression
and Purifi cation
of Amphiphysin

ʼ g/mL kanamycin.

3. Isopropyl-1-thio- - D -galactopyranoside (IPTG) is dissolved at

1 M in water and stored frozen.
4. HisTrapp FF column (1-mL cartridge; from GE Healthcare).
5. Sonication buffer: 50 mM Tris/HCl (pH 8), 300 mM NaCl,
10 % glycerol, and 10 mM imidazole. Wash and elution buffers
contain increased imidazole concentrations of 50, 75, 100,
150, 200, or 250 mM.
6. Tip sonicator such as a Branson Cell Disruptor B15.

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1. Synthetic peptides LY (amino acid sequence CRKRSHAGYQTI)
and LA (CRKRSHAGAQTI) correspond to the cytoplasmic
domain of Lamp1 (lysosome-associated membrane protein-1)
and a transport-defi cient mutant with the critical tyrosine
mutated to alanine, respectively. Peptides were purchased at
more than 70 % purity from, e.g., NeoMPS. The N-terminal
cysteines were added to the sequences for coupling.
2. Glass tubes resistant to liquid nitrogen, e.g., 12-cm test tubes
NS 14.5/23 for vacuum from Glas Keller.
3. Nucleopore polycarbonate membrane with 400-nm pore size
and prefi lters (drain disc 10 mm PE) from Whatman.
4. Mini-Extruder from Avanti Polar Lipids https://www.avan-
tilipids.com/index.php?option=com_content&view=article&i
d=185&Itemid=193 .
5. Egg L -

2.5 Preparation
of Liposomes
and Peptidoliposomes

-phosphatidylcholine (PC) from Avanti Polar Lipids.
6. Soybean L -

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-phosphatidylcholine, also called azolectin
(Sigma). This is a mixture of phospholipids, containing only
20 % PC, however.
7. MMCC-DOPE ( N -[{4-maleimidylmethyl}cyclohexane-1-
carbonyl]-1,2-dioleoyl-sn-glycero-3-phosphoethanolamine)
from Avanti Polar Lipids is dissolved in chloroform/methanol
2:1 (v/v). Aliquots of 125 nmol are dried in silanized 1.5-mL
reaction tubes and stored at −20 °C.
8. Chloroform/methanol 2:1 (v/v).
9. 1,1-Dichloromethane.
10. Liposome buffer: 10 mM HEPES-NaOH, pH 6.5, 100 mM
NaCl, 0.5 mM EDTA.

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