Biology Reference
In-Depth Information
3
Methods
3.1 In Vitro
Transcription of mRNA
from Plasmid DNA
In the following Subheadings
3.1
-
3.7
, liposome-supplemented
WGCF system for integral MP production is described. This
method is optimized for the synthesis of functional AtDTC pro-
teins. The typical yield of MPs produced in the bilayer method [
4
]
described here is usually 1-10
l reaction [
5
]. These
amounts are suffi cient for functional analysis. The transport assay
of AtDTC is also described.
μ
g/150
μ
1. Prepare 50
μ
l of transcription mixture as follows: 10
μ
l of 5×
transcription buffer, 6
μ
l of 25 mM NTP mix, 5
μ
l of template
plasmid (1
μ
g/
μ
l), 0.5
μ
l of RNase inhibitor, 0.625
μ
l of SP6
RNA polymerase, and 27.875
μ
l of Milli-Q water.
2. Incubate at 37 °C for 3-6 h.
3. Centrifuge the transcription mixture at 20,000 ×
g
for 1 min.
4. Transfer the supernatant containing the mRNA for translation
to new centrifuge tube (
see
Note 2
).
3.2 Preparation
of Liposomes
1. Dissolve 10 g of asolectin in 30 ml of chloroform.
2. Add 180 ml of ice-cold acetone to the solution and stir the
suspension on a magnetic stirrer for 2 h at room temperature.
3. Turn off the stirrer and allow the solution to stand overnight
at 4 °C in order to precipitate phospholipids.
4. Aspirate the supernatant as much as possible using a glass
pipette and dry the pellet completely under a fl ow of nitrogen
gas.
5. Store the dried phospholipid mixture at −20 °C until use.
6. Prepare the liposome suspension at 5 mg/ml in Milli-Q water.
7. Sonicate the liposome suspension (10 % amplitude and 30 %
duty cycle) on ice until the appearance changes from milky to
nearly transparent.
8. Prepare unilamellar liposomes by extrusion through a mini
extruder. To ensure proper sizing, pass the liposome suspen-
sion 11 times through a 0.4
μ
m polycarbonate membrane each
followed by a 0.1
μ
m membrane (
see
Note 3
).
3.3 Bilayer Synthesis
of AtDTC Proteins
by Liposome-
Supplemented
WGCF System
1. Thaw WG extract (WEPRO 1240), creatine kinase, and dialy-
sis buffer on ice. Keep all reagents on ice while in use. Freeze
promptly afterwards. Prepare 25
μ
l of translation mixture on
ice as follows: 6.25
μ
l of WG extract, 0.25
μ
l of 40 mg/ml
creatine kinase, 4.7
μ
l of 4× dialysis buffer, 2.5
μ
l of 5 mg/ml
liposomes, 7.5
μ
l of mRNA, and 3.8
μ
l of Milli-Q water.
2. Prepare 125
μ
l of substrate mixture on ice as follows: 31.25
μ
l
of 4× dialysis buffer, 93.75
μ
l of Milli-Q water.
