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In Vitro Translation: The Basics
•The in vitro synthesis of proteins in cellfree extracts is an important tool for
molecular biologists.
•It has a variety of applications,
including the Optimization of protien
expression, localization of mutations
through synthesis of truncated gene
products, protein folding studies etc.
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).
These immature red cells, 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.
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.
In eukaryotes, the Kozak sequence A/GCCACCAUGG, which
lies within a short 5' untranslated region, directs translation
of mRNA..
In contrast to the E. coli ribosome, which preferentially
recognizes the Shine-Dalgarno sequence
.
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.
Trichloro acetic acid for protien precipitation
Standard in Vitro Translation
Procedure Using Rabbit Reticulocyte
Lysate
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
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
•Many eukaryotic proteins insoluble upon
expression
•Eukaryotic co- and post-translational
modifications not possible
•Codon usage is different from eukaryotes
•Mammalian system
•Modifications are possible
•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)
•Lower yields than E. coli
•New system
E. coli
Rabbit Reticulocyte (RRL)
Wheat germ
Insect
Human