Transcript IN VITRO TRANSCRIPTION . TRANSLATION - e

In Vitro Translation: The Basics
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.
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.
Cell-Free Expression Systems
The most frequently used cell-free translation systems consist of extracts from
rabbit reticulocytes 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.
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.
The Kit 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.
Exogenous proteins are synthesized at a rate close to that observed in intact
reticulocyte cells.
Standard in Vitro Translation
Procedure Using Rabbit Reticulocyte
Lysate
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).
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
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
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
Phage Polymerase Promoters: Minimal Sequence
Requirements
The bacteriophage promoters, T7, T3, and SP6, consist of 23 basepairs numbered -17
to +6, where +1 indicates the first base of the coded transcript. An important observation
is that, of the +1 through +6 bases, only the base composition of +1 and +2 are critical
and must be a G and purine, respectively, to yield an efficient transcription template. In
addition, synthetic oligonucleotide templates only need to be double-stranded in the -17
to -1 region of the promoter, and the coding region can be all single-stranded. This can
reduce the cost of synthetic templates, since the coding region (i.e., from +1 on) can be
left single-stranded, and the short oligonucleotides required to render the promoter
region double-stranded can be used with multiple templates.
Consensus Promoter Sequences. The +1
base (in bold) is the first base incorporated into
RNA during transcription. The underline
indicates the minimum sequence required for
efficient transcription.
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. 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.
Linked in Vitro Transcription and
Translation Procedure Using Rabbit
Reticulocyte Lysate
Human In Vitro Translation
Human In Vitro Protein Expression System is a method for expressing proteins
from DNA or mRNA templates in a cell-free solution containing essential
components of the cellular translational machinery. Extracts of an immortalized
human cell line provide the ribosomes, initiation and elongation factors, tRNAs
and other basic components required for protein synthesis. When supplemented
with proprietary accessory proteins, ATP, and an energy regenerating system,
these extracts sustain the synthesis of target proteins from DNA templates for up
to 6 hours without the need to remove inhibitory byproducts.
Protein synthesis begins with either a DNA or RNA template. When starting with
DNA, mRNA is transcribed in one hour at 32°C using the included reaction
components. The mRNA are then added to the in vitro translation reaction and
incubated for 90 minutes at 30°C for general protein expression. Alternatively, the
reaction is incubated for 90 minutes at 28°C for glycoprotein expression.
Glycoprotein Expression Kits
The major advantage of using the Human In Vitro Expression System is the ability
to generate protein modifications just as they occur in vivo. The Glycoprotein
Expression Kits are optimized for generating glycosylated protein. The yield of
glycoprotein produced in the system out-performs traditional methods (i.e., those
based on rabbit reticulocyte lysates combined with microsomal membranes) and
have higher fidelity than insect expression systems, which lack N-linked
oligosaccharide side chains
Human choriogonadotropin
hormone (hCG) β subunit was
expressed in insect cell
extract or the Pierce System
according to the
manufacturer's instructions.
Insect lysates yielded no
glycosylated protein when
1µg of hCGβ mRNA was used
and multiple glycoforms when
12µg of mRNA was used. Note
that the hCGβ was cloned into
vectors that were
recommended by the
manufacturer of the insect
expression system
Traditional in vitro translation systems have limitations in the types of proteins
that can be generated.
The Human in vitro Translation System uses human HeLa or hybridoma lysates
that endogenously contain the enzymes required for protein synthesis and proper
post-translational modifications.
The types of proteins that can be expressed using the Human in vitro Translation
System, include:
Proteins large and small
Phosphoproteins
Glycoproteins
Membrane proteins
Successful protein expression is influenced by protein stability, folding and
solubility. High molecular weight proteins are difficult to express in vitro because
of the greater chance of premature termination and the greater complexity of the
tertiary structure compared to smaller proteins, which increases the chance of
improper folding . Additionally, proteins with greater size can have large
hydrophobic regions that promote the formation of inclusion bodies when
expressed in E. coli extracts.
The Human in vitro Translation System can be used to produce a broad range of
proteins of different sizes.
Size distribution of proteins generated with the Human in vitro Translation
System. One hundred proteins ranging from 8kDa to greater than 250kDa were
individually expressed using the Pierce Human in vitro Translation System with a 95%
success rate.
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).
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 ShineDalgarno ribosome binding site upstream of the initiator codon.
Capping of eukaryotic RNA is not required.
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 purinerich 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).
Ribosomal Binding Site Sequence Requirements
Protein synthesis is regulated by the sequence and structure of the 5'
untranslated region (UTR) of the mRNA transcript. In prokaryotes, the ribosome
binding site (RBS), which promotes efficient and accurate translation of mRNA, is
called the Shine-Dalgarno sequence. This purine-rich sequence of 5' UTR is
complementary to the UCCU core sequence of the 3'-end of 16S rRNA (located
within the 30S small ribosomal subunit). Various Shine-Dalgarno sequences have
been found in prokaryotic mRNAs.
These sequences lie about 10 nucleotides upstream from the AUG start codon.
Activity of a RBS can be influenced by the length and nucleotide composition of
the spacer separating the RBS and the initiator AUG.
In eukaryotes, the Kozak sequence A/GCCACCAUGG, which lies within a short 5'
untranslated region, directs translation of mRNA. An mRNA lacking the Kozak
consensus sequence may be translated efficiently in vitro systems if it possesses
a moderately long 5' UTR that lacks stable secondary structure.
In contrast to the E. coli ribosome, which preferentially recognizes the ShineDalgarno sequence, eukaryotic ribosomes (such as those found in retic lysate)
can efficiently use either the Shine-Dalgarno or the Kozak ribosomal binding sites.
.
The +1 A is the first base of the
AUG initiator codon (shaded)
responsible for binding of fMettRNAfMet. The underline
indicates the ribosomal binding
site sequence, which is required
for efficient translation.
Comparison of in vitro (cell-free) protein expression systems. Advantages and disadvantages of existing extract-based systems
for human recombinant protein synthesis. Selection of a cell-free expression system should consider the biological nature of the
protein, application, and the template used for protein expression.
System
Advantages
Disadvantages
•Very high protein yield
•Relatively tolerant of additives
•Many eukaryotic proteins insoluble upon
expression
•Eukaryotic co- and post-translational
modifications not possible
•Codon usage is different from eukaryotes
•Mammalian system
•Cap independent translation
•Sensitive to additives
•Protein glycosylation not possible
•Co-expression of off-target proteins
•Translation of large proteins possible
•Devoid of off-target endogenous
mammalian proteins
•High protein yield
•Mammalian co- and post-translational
modifications are not possible
•Premature termination of products
•Translation of large proteins possible
•No endogenous mammalian proteins
•Certain forms of protein glycosylation
possible
•Non-mammalian
•Human system
•Co- and post-translational modifications
are possible
•Synthesis of functional proteins
•Possible to make VPLs (virus-like
particles)
•Sensitive to additives
•Lower yields than E. coli
•New system
E. coli
Rabbit Reticulocyte (RRL)
Wheat germ
Insect
Human