Biology Reference
In-Depth Information
WGCF synthesis and purifi cation of properly folded bacteriorho-
dopsin (bR), which is described below [
3
].
Lastly, we describe method for WGCF synthesis of peripheral
MP modifi ed by
N
-myristoylation (Fig.
1c
). In the optimized con-
dition reported here,
N
-myristoylated proteins produced by
WGCF system are membrane bound.
2
Materials
1.
Ex Taq
DNA polymerase (5 units/μl)
μ
l) and 10×
Ex Taq
buffer
2.1 Preparation
of mRNA
(Takara, Shiga, Japan).
2. 2.5 mM dNTP mixture: 2.5 mM each of dATP, dCTP, dGTP,
and dTTP.
3. 5× transcription buffer: 400 mM HEPES-KOH, pH 7.8,
80 mM magnesium acetate, 10 mM spermidine, 50 mM DTT.
4. 25 mM NTP mixture: 25 mM each of ATP, CTP, GTP, and
UTP.
5. Ribonuclease (RNase) inhibitor (80 units/μl)
μ
l) and SP6 RNA
polymerase (80 units/μl)
μ
l) (Promega, Madison, WI).
1. Soybean asolectin (Fluka, Buchs, Switzerland).
2. Chloroform and acetone (Nacalai tesque, Kyoto, Japan).
3. Sonicator (Digital Sonifi er model 250 D, 200 W, 20 kHz)
(Branson, Danbury, CT).
4. Mini extruder (Avanti Polar Lipids, Alabaster, AL).
2.2 Preparation
of Liposomes
2.3 WGCF Protein
Synthesis
1. WG extract (WEPRO1240, OD
260
= 240, and WEPRO7240H,
OD
260
= 240) (CellFree Science Co. Ltd., Matsuyama, Japan).
2. Creatine kinase (Roche Diagnostics K. K., Tokyo, Japan) is
dissolved in water at 40 mg/ml and stored in aliquots at
−80 °C.
3. Dialysis buffer (4×): 120 mM HEPES-KOH (pH 7.8),
400 mM potassium acetate, 10.8 mM magnesium acetate,
1.6 mM spermidine, 10 mM dithiothreitol, 4.8 mM ATP,
1 mM GTP, 64 mM creatine phosphate, 0.02 % NaN
3
, and
1.2 mM of each amino acid. Store at −80 °C (
see
Note 1
).
4. Fos-choline 14 (FC14) (Anatrace, Maumee, OH).
5. 3-[(3-Cholamidopropyl)dimethylammonio]-1-
propanesulfonate (CHAPS) (Nacalai tesque).
6. FC14/CHAPS mixture: 25 mM FC14 and 37.5 mM CHAPS.
7. 100 mM retinal: Dissolve 28.4 mg of all-trans retinal (Wako
Pure Chemical, Osaka, Japan) in 1 ml dimethyl sulfoxide
(sterile-fi ltered, Nacalai tesque). Store at −20 °C.
