Protocols and Applications Guide (US Letter Size) - Promega

||||| 5Protein Expression
Coupled Systems, since the master mix contains all amino
acids except methionine, and the labeling efficiency with
other amino acids will be significantly reduced.
Table 5.3. Recommended Concentrations of Alternative
Radiolabeled Amino Acids.
Final
Concentration Volume
Amino Acid
in Reaction Required
[3H]leucine
(100–200Ci/mmol)
0.5mCi/ml
5µl
[14C]leucine
(300mCi/mmol)
[35S]cysteine
(1,200Ci/mmol)
5µCi/ml
0.3mCi/ml
5µl
5µl
[35S] Methionine
We recommend using a “translational grade”
[35S]methionine such as PerkinElmer EasyTag
L-[35S]methionine (PerkinElmer Cat.# NEG709A). We have
obtained acceptable results using 1–4µl of [35S]methionine
(1,200Ci/mmol at 10mCi/ml). Depending upon the
translational efficiency of the experimental RNA/DNA and
number of methionines present in the protein, the amount
of [35S]methionine can be adjusted to balance exposure
time against label cost. When using and storing
[35S]methionine, follow the manufacturer’s
recommendations.
Non-Radioactive Protein Labeling
The Transcend Non-Radioactive Translation Detection
Systems (Cat.# L5070 and L5080) and the FluoroTect
GreenLys in vitro Translation Labeling System (Cat.# L5001)
can be used with any of the TNT® Coupled or Quick
Coupled Systems. These systems use a precharged lysine
tRNA, which is incorporated into the translated protein.
The Transcend System incorporates a biotinylated lysine,
which can be detected by blotting and probing with
streptavidin/AP or streptavidin HRP. The FluoroTect
Reagent incorporates a fluorescently labeled lysine
(BODIPY®), which can be detected directly in the gel.
III. Eukaryotic Cell-Free Expression Systems
The TNT® Coupled Reticulocyte Lysate Systems offer
researchers an alternative for eukaryotic in vitro
transcription and translation: a one-tube, coupled
transcription/translation system. Standard Rabbit
Reticulocyte Lysate or Wheat Germ Extract translations
(Pelham and Jackson, 1976) commonly use RNA
synthesized in vitro (Krieg and Melton, 1984) from SP6, T3
or T7 RNA polymerase promoters. The RNA is then used
as a template for translation. The TNT® Systems bypass
many of these steps by incorporating transcription directly
in the translation mix.
In most cases, the TNT® System reactions produce
significantly more protein (two- to sixfold) in a 1- to 2-hour
reaction than standard in vitro Rabbit Reticulocyte Lysate
Protocols & Applications Guide
www.promega.com
rev. 6/09
or Wheat Germ Extract translations using RNA templates.
In addition, TNT® Lysates also can be used with microsomal
membranes to study processing events (see Section VII).
A. TNT® SP6 High-Yield Protein Expression System
The TNT® SP6 High-Yield Protein Expression System uses
a high-activity wheat germ extract supplemented with SP6
RNA polymerase and other components to express
10–100µg/ml of soluble protein in a one-tube coupled
transcription/translation format. The system produces
substantially more protein than conventional wheat germ
systems while maintaining the ease of use that cell-free and
coupled transcription and translation conditions afford.
Following a two-hour incubation, the expressed protein
can be used directly in downstream applications; no protein
purification steps are necessary. For a detailed protocol
and background information on this system, please see
Technical Manual #TM282 (www.promega.com
/tbs/tm282/tm282.html).
B. TNT® T7 Insect Cell Extract Protein Expression System
The TNT® T7 Insect Cell Extract Protein Expression System
is a single-tube, coupled transcription/translation system
for the cell-free expression of proteins. The extract is made
from the commonly used Spodoptera frugiperda Sf21 cell line
(Ezure et al. 2006) However, the extract contains no
endogenous glycosylation machinery. All necessary
components are present in the TNT® T7 ICE Master Mix
(ICE = insect cell extract). Protein is expressed from genes
cloned downstream of the T7 polymerase promoter. The
best expression will be achieved using vectors that contain
the baculovirus polyhedrin 5´ and 3´ untranslated region
(Suzuki et al. 2006). Two vectors, the pF25A and pF25K ICE
T7 Flexi® Vectors (Cat.# L1061 and L1081), have been
designed specifically to produce optimal protein yields in
this system. For a detailed protocol and background
information on this system, please see Technical Manual
#TM305 (www.promega.com/tbs/tm305/tm305.html).
The pF25A and pF25K ICE T7 Flexi® Vectors are compatible
with the Flexi® Cloning System (Cat.# C8640), which allows
easy transfer of sequences to and from additional Flexi®
Vectors. Additional Flexi® Vectors are available for use
with other protein expression systems. For a detailed
protocol and background information on this system, please
see Technical Manual #TM254 (www.promega.com
/tbs/tm254/tm254.html).
C. TNT® Coupled Wheat Germ Extract Systems—Coupled
Transcription/Translation
The TNT® Wheat Germ Extract Systems are available in
five configurations for transcription and translation of genes
cloned downstream from the SP6, T3 or T7 RNA
polymerase promoter. With these systems, a 50µl reaction
is programmed with 0.2–2µg of template and incubated
for 1.5 hours at 30°C. For a detailed protocol and
background information about this system, please see
Technical Bulletin #TB165 (www.promega.com
/tbs/tb165/tb165.html).
PROTOCOLS & APPLICATIONS GUIDE 5-5