Báo cáo khoa học: Cell-free protein synthesis ppt

MINIREVIEW SERIES
Cell-free protein synthesis
Nicholas E. Dixon
Research School of Chemistry, Australian National University, Canberra, ACT, Australia
Cell-free protein synthesis using cell extracts from
Escherichia coli, wheat germ and rabbit reticulocytes
has been used for over 40 years to produce small
amounts of radiolabeled proteins for identification of
gene products and other applications. In the E. coli
system programmed with plasmid DNA, the cell
extract contains or is supplemented with an RNA po-
lymerase to transcribe the gene, and the mRNAs are
translated by a complex mixture that contains ribo-
somes and a full complement of initiation, elongation
and termination factors, as well as a full set of amino-
acyl-tRNA synthetases and other required enzymes.
The presence of molecular chaperones, protein disul-
fide and peptidyl-prolyl cis-trans isomerases generally
ensures that proteins are correctly folded into soluble,
active forms. With the eukaryotic extracts, it has addi-
tionally been possible to program protein synthesis
directly with in vitro transcribed mRNA that can be
produced directly from PCR products, and the pres-
ence of factors responsible for post-translational modi-
fication of proteins ensures the production of fully
functional gene products.
Given the simplicity of these systems, it is not sur-
prising that considerable effort has been invested over
the past two decades to improve their productivity and
efficiency. The key breakthroughs were the develop-
ment of methods for continuous feeding of amino

acids and nucleoside triphosphates into the reaction
mixtures, and the use of efficient energy-regeneration
systems. As a result, cell-free synthesis can now be
used routinely to rapidly make milligram quantities of
proteins for a range of applications, especially in struc-
tural and functional proteomics. Coupled with improve-
ments in high-throughput protein isolation by use of
purification tags, and the efficiency and sensitivity of
downstream applications like protein crystallization
and NMR spectroscopy, such quantities of proteins
are now adequate for complete structure determin-
ation. Especially exciting new developments are in the
use of selected lipids and detergents in cell-free
reactions to solubilize membrane proteins in fully
functional membrane-inserted forms.
That preparative cell-free protein production systems
can now be programmed not only by plasmid DNAs,
but also by PCR products and in vitro-transcribed
mRNAs, has enabled rapid isolation of useful quanti-
ties of proteins in high-throughput applications with-
out ever having to resort to use of living cells. It is
possible, starting with PCR products, to simulta-
neously generate large numbers of genes or mutant
versions of a single gene, express them in parallel, and
analyse their functions within periods of days rather
than months.
While maximization of protein production is clearly
a very useful outcome, it is not the only application of
these systems. As an example, direct screening for
genes that encode proteins with selected functions from

large gene libraries (e.g., in directed molecular evolu-
tion experiments or in functional genomics applica-
tions) might require production of assayable quantities
of an enzyme from a single copy of a gene in an
artificial compartment (e.g., a droplet). For such appli-
cations, attention has turned to the use of fully func-
tional transcription ⁄ translation systems reconstituted
with highly purified ribosomes, enzymes, and trans-
lation factors. The expectation is that these ‘pure’
systems will enable new technologies, and should result
in better understanding of the processes of translation
that can be exploited to further manipulate reactions
for better productivity in particular circumstances.
This series of minireviews examines four different
aspects of the use of cell-free translation systems. Each
article describes the current state of the art and high-
lights future prospects and challenges. In the first mini-
review, Shimizu et al. give an overview of the various
cell-free translation systems, and then highlight the
roles of the chaperones and folding isomerases in pro-
tein folding, and use of cell-free methods for capturing
membrane proteins in liposomes, for insertion of un-
natural amino acids into proteins, and for directed
molecular evolution experiments using their recently
developed fully reconstituted system.
The second minireview by Klammt et al. gives a
thorough overview of the use of cell-free systems for
doi: 10.1111/j.1742-4658.2006.05445.x
FEBS Journal 273 (2006) 4131–4132 ª 2006 The Author Journal compilation ª 2006 FEBS 4131
production of integral membrane proteins, both in

insoluble forms that are often easily resolubilized in
detergents and, in the presence of a range of deter-
gents, in soluble form. This article gives an excellent
guide to strategies for optimization of reactions with
such challenging proteins.
The next minireview by Ozawa et al. focuses on the
use of cell-free protein synthesis for direct isotopic
labeling of proteins for NMR applications. Because
only the newly synthesized protein is labeled, NMR
spectra can be run directly using the reaction mixture,
and combinatorial [
15
N]-labeling can be used to rapidly
assign peaks in HSQC spectra to a particular amino
acid type. This not only increases efficiency in NMR
structure determination, but simplifies study of pro-
tein–ligand interactions.
Finally, Vinarov et al. describe how an optimized
wheat germ system is being used in high-throughput
formats to produce proteins for NMR structure
determination as part of their eukaryotic structural
genomics program. Their successes show that cell-free
protein synthesis has come of age as an alternative
to cell-based methods for high-throughput applica-
tions.
Nick Dixon received his PhD training in Biochemistry at the University of Queensland with Burt Zerner, and
followed this with postdoctoral appointments in Chemistry with Alan Sargeson at the Australian National
University (ANU) and in Biochemistry with Arthur Kornberg at Stanford. He established his research group at
the ANU in 1986, where he is currently Professor of Biological Chemistry. His research focuses on the bac-
terial DNA replication machinery as a model system to study the chemistry of protein–protein interactions in

dynamic macromolecular machines.
Cell-free protein synthesis N. E. Dixon
4132 FEBS Journal 273 (2006) 4131–4132 ª 2006 The Author Journal compilation ª 2006 FEBS