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The in vitro synthesis of proteins in
cell-free extracts is an important tool for molecular biologists
and has a variety of applications, including the rapid
identification of gene products (e.g., proteomics), localization
of mutations through synthesis of truncated gene products,
protein folding studies, and incorporation of modified
or unnatural amino acids for functional studies. The use
of in vitro translation systems can have advantages over
in vivo gene expression when the over-expressed product
is toxic to the host cell, when the product is insoluble
or forms inclusion bodies, or when the protein undergoes
rapid proteolytic degradation by intracellular proteases.
In principle, it should be possible to prepare a cell-free
extract for in vitro translation of mRNAs from any type
of cells. In practice, only a few cell-free systems have
been developed for in vitro protein synthesis. In general,
these systems are derived from cells engaged in a high
rate of protein synthesis. This article will explain different
approaches to in vitro protein synthesis (translation of
purified RNA versus "linked" and "coupled" transcription:translation)
and will also describe basic differences between eukaryotic
and prokaryotic cell-free systems.
Cell-Free Expression Systems
The most frequently used cell-free
translation systems consist of extracts from rabbit reticulocytes,
wheat germ and Escherichia coli. All are prepared
as crude extracts containing all the macromolecular components
(70S or 80S ribosomes, tRNAs, aminoacyl-tRNA synthetases,
initiation, elongation and termination factors, etc.)
required for translation of exogenous RNA. To ensure
efficient translation, each extract must be supplemented
with amino acids, energy sources (ATP, GTP), energy regenerating
systems (creatine phosphate and creatine phosphokinase
for eukaryotic systems, and phosphoenol pyruvate and
pyruvate kinase for the E. coli lysate), and other
co-factors (Mg2+, K+,
etc.).
There are two approaches to in
vitro protein synthesis based on the starting genetic
material: RNA or DNA. Standard translation systems, such
as reticulocyte lysates and wheat germ extracts, use
RNA as a template; whereas "coupled" and "linked" systems
start with DNA templates, which are transcribed into
RNA then translated. Each of these systems is discussed
below. In addition, a summary of Ambion's translation
systems can be found in the following comparison
chart.
Translation Systems
Rabbit Reticulocyte Lysate
Rabbit reticulocyte lysate is
a highly efficient in vitro eukaryotic protein synthesis
system used for translation of exogenous RNAs (either
natural or generated in vitro). In vivo, reticulocytes
are highly specialized cells primarily responsible for
the synthesis of hemoglobin, which represents more than
90% of the protein made in the reticulocyte. These immature
red cells have already lost their nuclei, but contain
adequate mRNA, as well as complete translation machinery,
for extensive globin synthesis. The endogenous globin
mRNA can be eliminated by incubation with Ca2+-dependent
micrococcal nuclease, which is later inactivated by chelation
of the Ca2+ by EGTA.
Ambion offers a nuclease-treated reticulocyte
lysate.
This type of lysate is the most widely used RNA-dependent
cell-free system because of its low background and its
efficient utilization of exogenous RNAs even at low concentrations
(Figure 1). Exogenous proteins are synthesized at a rate
close to that observed in intact reticulocyte cells.
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| Figure 1. Standard
in Vitro Translation Procedure Using Rabbit Reticulocyte
Lysate or Wheat Germ Extract. |
Untreated reticulocyte lysate translates
endogenous globin mRNA, exogenous RNAs, or both. This type
of lysate is typically used for studying the translation
machinery, e.g. studying the effects of inhibitors on globin
translation. Both the untreated and treated rabbit reticulocyte
lysates have low nuclease activity and are capable of synthesizing
a large amount of full-length product. Both lysates are
appropriate for the synthesis of larger proteins from either
capped or uncapped RNAs (eukaryotic or viral).
Wheat Germ Extract
Wheat germ extract is a convenient
alternative to the rabbit reticulocyte lysate cell-free
system. This extract has low background incorporation
due to its low level of endogenous mRNA. Wheat germ lysate
efficiently translates exogenous RNA from a variety of
different organisms, from viruses and yeast to higher
plants and mammals. The wheat germ extract is recommended
for translation of RNA containing small fragments of
double-stranded RNA or oxidized thiols, which are inhibitory
to the rabbit reticulocyte lysate. Both retic and wheat
germ extracts translate RNA isolated from cells and tissue
or those generated by in vitro transcription (see Figure
1). When using RNA synthesized in vitro, the presence
of a 5' cap structure may enhance translational activity.
Typically, translation by wheat germ extracts is more
cap-dependent than translation by retic extracts. If
capping of the RNA is impossible and the protein yield
from an uncapped mRNA is low, the coding sequence can
be subcloned into a prokaryotic vector and expressed
directly from a DNA template in an E.coli cell-free
system.
E. coli Cell-Free
System
E. coli cell-free
systems consist of a crude extract that is rich in endogenous
mRNA. The extract is incubated during preparation so that
this endogenous mRNA is translated and subsequently degraded.
Because the levels of endogenous mRNA in the prepared lysate
is low, the exogenous product is easily identified. In
comparison to eukaryotic systems, the E.coli extract
has a relatively simple translational apparatus with less
complicated control at the initiation level, allowing this
system to be very efficient in protein synthesis. Bacterial
extracts are often unsuitable for translation of RNA, because
exogenous RNA is rapidly degraded by endogenous nucleases.
There are some viral mRNAs (TMV, STNV, and MS2) that translate
efficiently, because they are somewhat resistant to nuclease
activity and contain stable secondary structure. However, E.coli extracts
are ideal for coupled transcription:translation from DNA
templates.
"Linked" And "Coupled" Transcription:Translation
Systems
In standard translation reactions,
purified RNA is used as a template for translation. "Linked" and "coupled" systems,
on the other hand, use DNA as a template. RNA is transcribed
from the DNA and subsequently translated without any
purification. Such systems typically combine a prokaryotic
phage RNA polymerase and promoter (T7, T3, or SP6) with
eukaryotic or prokaryotic extracts to synthesize proteins
from exogenous DNA templates. DNA templates for transcription:translation
reactions may be cloned into plasmid vectors or generated
by PCR (Primer Sequences for PCR-generated
Translation Templates).
Linked Transcription:Translation
The "linked" system is a
two-step reaction, based on transcription with a bacteriophage
polymerase followed by translation in the rabbit reticulocyte
lysate or wheat germ lysate (Figure 2). Because the transcription
and translation reactions are separate, each can be optimized
to ensure that both are functioning at their full potential.
Conversely, many commercially available eukaryotic coupled
transcription:translation systems have compromised one
or both reactions so that they can occur in a single tube.
Thus, yield is sacrificed for convenience.
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| Figure 2. Linked
in Vitro Transcription and Translation Procedure
Using Rabbit Reticulocyte Lysate. |
Coupled
Transcription:Translation
Unlike eukaryotic systems where transcription and translation occur sequentially,
in E. coli, transcription and translation occur simultaneously within
the cell. In vitro E. coli translation systems are thus performed the
same way, coupled, in the same tube under the same reaction conditions (one-step
reaction; Figure 3). During transcription, the 5' end of the RNA becomes available
for ribosomal binding and undergoes translation while its 3' end is still being
transcribed. This early binding of ribosomes to the RNA maintains transcript
stability and promotes efficient translation. This bacterial translation system
gives efficient expression of either prokaryotic or eukaryotic gene products
in a short amount of time. For the highest protein yield and the best initiation
fidelity, make sure the DNA template has a Shine-Dalgarno ribosome binding
site upstream of the initiator codon. Capping of eukaryotic RNA is not required.
Use of E.coli extract also eliminates cross-reactivity or other problems
associated with endogenous proteins in eukaryotic lysates. Also, the E.
coli S30 extract system allows expression from DNA vectors containing natural E.
coli promoter sequences (such as lac or tac).
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| Figure 3. Coupled
in Vitro Transcription:Translation Procedure Using E.
coli Extract. |
Important Elements For Translation
There are some significant differences
between prokaryotic and eukaryotic mRNA transcripts. Typically,
eukaryotic mRNAs are characterized by two post-transcriptional
modifications: a 5'-7 methyl-GTP cap and a 3' poly(A) tail.
Both modifications contribute to the stability of the mRNA
by preventing degradation. Additionally, the 5' cap structure
enhances the translation of mRNA by helping to bind the
eukaryotic ribosome and assuring recognition of the proper
AUG initiator codon. This function may vary with the translation
system and with the specific mRNA being synthesized. The
consensus sequence 5'-GCCACCAUGG-3', also known as the "Kozak" sequence,
is considered to be the strongest ribosomal binding signal
in eukaryotic mRNA. For efficient translation initiation,
the key elements are the G residue at the +1 position and
the A residue at the -3 position. An mRNA that lacks the
Kozak consensus sequence may be translated efficiently
in eukaryotic cell-free systems if it possesses a moderately
long 5'-untranslated region (UTR) that lacks stable secondary
structure.
In bacteria, the ribosome is guided to
the AUG initiation site by a purine-rich region called
the Shine-Dalgarno (SD) sequence. This sequence is complementary
to the 3' end of the 16s rRNA in the 30S ribosomal subunit.
Upstream from the initiation AUG codon, the SD region has
the consensus sequence 5'-UAAGGAGGUGA-3'. Specific mRNAs
vary considerably in the number of nucleotides that complement
the anti-Shine-Dalgarno sequence of 16S rRNA, ranging from
as few as two to nine or more. The position of the ribosome
binding site (RBS) in relation to the AUG initiator is
very important for efficiency of translation (usually from
-6 to -10 relative to the A of the initiation site).
See "Ribosomal
Binding Sites Sequence Requirements" for more
information.
Products For In Vitro Translation
Ambion offers both eukaryotic- and prokaryotic-derived
cell-free translation systems.
Retic
Lysate IVT™ Kit:
For the efficient translation of in vitro synthesized
transcripts, poly(A) and total RNA.
PROTEINscript™ II:
Linked transcription:translation kit for efficient in vitro
synthesis of proteins (eukaryotic or prokaryotic) directly
from PCR products, or supercoiled or linear DNA templates
containing a T7 polymerase promoter.
Primer Sequences
for PCR-generated Translation Templates
DNA templates for translation using "coupled" or "linked" transcription:translation
systems can be easily generated by PCR. Below are the
upstream (5')primer sequences to produce PCR products
for T7-driven transcription and subsequent translation
in a retic lysate and E.coli extract, respectively.
Note, translation systems that use T3 or SP6 polymerases
are also available . To generate PCR templates for other
polymerases, simply change the T7 Promoter Sequence to
a T3
or an SP6 sequence.
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