Protocols and Applications Guide (US Letter Size) - Promega

||||| 5Protein Expression
TNT ® Master Mix
(Coupled
Transcription/
Translation)
MRNA
Figure 5.1. Cell-Free expression using the TNT® systems.
A. mRNA-Driven Translation
The Rabbit Reticulocyte Lysate Translation Systems
(Nuclease-treated and Untreated), Flexi® Rabbit
Reticulocyte Lysate System and Wheat Germ Extract System
are all used for translation of mRNA. Table 5.1 summarizes
these systems.
The Rabbit Reticulocyte Lysate, Nuclease-Treated, and the
Flexi® Rabbit Reticulocyte Lysate have been optimized for
mRNA translation by adding several supplements. These
include hemin, which prevents activation of the
heme-regulated eIF-2a kinase (HRI); an energy-generating
system consisting of pretested phosphocreatine kinase and
phosphocreatine; and calf liver tRNAs, to balance the
accepting tRNA populations, thus optimizing codon usage
and expanding the range of mRNAs that can be translated
efficiently. In addition both lysates are treated with
micrococcal nuclease to eliminate endogenous mRNA, thus
reducing background translation. The Flexi® Rabbit
Reticulocyte Lysate System provides greater flexibility of
reaction conditions than the Rabbit Reticulocyte Lysate,
Nuclease-Treated, by allowing translation reactions to be
optimized for a wide range of parameters, including Mg2+
and K+ concentrations, and offers the choice of adding DTT.
In contrast, the Rabbit Reticulocyte Lysate, Untreated,
contains the cellular components necessary for protein
Table 5.1. Summary of mRNA-Driven Translation Systems.
System
Rabbit Reticulocyte Lysate,
Nuclease-Treated (Cat.#
L4960)
Rabbit Reticulocyte Lysate,
Untreated (Cat.# L4151)
Flexi® Rabbit Reticulocyte
Lysate (Cat.# L4540)
Wheat Germ Extract (Cat.#
L4380)
Protocols & Applications Guide
www.promega.com
rev. 6/09
Micrococcal
Nuclease-Treated
X
–
X
X
Untreated
–
X
–
–
Plasmid DNA
PCR Fragments
Gene sequence of
protein of interest
protein
NH 3
synthesis (tRNA, ribosomes, amino acids, initiation,
elongation and termination factors) but has not been treated
with micrococcal nuclease. Untreated Rabbit Reticulocyte
Lysate is used primarily for the isolation of translation
components, as an abundant source of endogenous globin
mRNA and to study protein synthesis of the endogenous
globin mRNA. Untreated Rabbit Reticulocyte Lysate is not
recommended for in vitro translation of specific mRNAs.
Wheat Germ Extract contains the cellular components
necessary for protein synthesis (tRNA, ribosomes, initiation,
elongation and termination factors). The extract is
optimized further by the addition of an energy-generating
system consisting of phosphocreatine and phosphocreatine
kinase, spermidine to stimulate the efficiency of chain
elongation and thus overcome premature termination, and
magnesium acetate at a concentration recommended for
the translation of most mRNA species. Only the addition
of exogenous amino acids (including an appropriately
labeled amino acid) and mRNA are necessary to stimulate
translation. Finally, Potassium Acetate is supplied as a
separate component so that the translational system may
be optimized for a wide range of mRNAs.
Salt
Optimization
Necessary
–
–
X
–
6634MA
Protein Yield in 50µl
Reaction
50–200ng
endogenous mRNA
synthesized
50–200ng
30–150ng
PROTOCOLS & APPLICATIONS GUIDE 5-2