Cell free translation

Molecular and Cellular Genetics WS 2008 / 2009
Cell free translation
Dr. Birgitta Beatrix
(AG Beckmann)
Pre-history of Cell-Free
Translation Systems
• 
1950s: protein synthesis does not
require integrity of the cell (H.
Borsook, T. Winnick, Greenberg)
• 
Early 1950s: Isolated fractions are
capable of synthesizing proteins
• 
1955: Ribonucleoprotein particles
identified in cells (G. Palade)
George E. Palade
(together with Albert Claude and Christian de Duve)
Nobel prize in Physiology and Medicine 1974
“for their discoveries concerning the structural
and functional organization of the cell”
Emeritus at UC San Diego
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Pre-history of Cell-Free
Translation Systems
•  Late 1950s: Isolation and physico chemical characterization of
Ribonucleoprotein particles
•  1958: Richard B. Roberts proposes the term “Ribosome” during
a symposium
Prediction of existence of mRNA
•  Alexander Spirin and Andrey Belozersky predicted the existence of
mRNA in the late 1950s
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Introduction of exogeneous
message
Marshall W. Nirenberg was awarded the Nobel Prize
in Physiology or Medicine in 1968 together with
Har Gobind Khorana and Robert W. Holley for
their interpretation of the genetic code and
its function in protein synthesis
Why cell free translation ??
•  Expression of toxic products
•  Incorporation of labelled isotops
•  Incorporation of modified amino acids
•  High through put applications
•  Protein folding studies
•  Protein labeling for structural studies (X-ray, NMR)
•  Import assays (mitochondria, chloroplasts, nucleus)
•  Preparation of translation intermediates (RNCs)
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In vitro Translation systems
Only a few systems are well established
•  E. coli
•  Yeast (S. cerevisiae)
•  Wheat germ
•  Rabbit reticulozyte lysate
But in principle every cell type should work
It all depends on optimizing the conditions
Prokaryotic versus Eukaryotic
•  prokaryotic
–  transcription and
translation in the same
reaction
–  plasmid, PCR fragment or
mRNA as template
–  T7 polymerase (SP6, T3 or
•  eukaryotic
–  in vitro transcription
separately
–  capping of mRNA
–  polyadenylation of mRNA
–  then translation
endogenous E. coli
–  processing of mRNA ?
polymerase)
–  protein modifications ?
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Preparation of cell free extracts
• 
is fairly easy
• 
grow cells to log phase (yeast, E. coli)
• 
quick cell disruption
• 
centrifugation to eliminate cell debris, organells, membranes,
aggregates
• 
gelfiltration to eliminate low molecular weight components
• 
RNase treatment
• 
aliquot and freeze in liquid N2
• 
optimize Mg2+, K+ concentration
• 
run-off translation (endogenous mRNA, yeast, E. coli)
How to get high yields ?
•  Problem: typically only 2-3 polypeptide molecules were
produced per mRNA molecule
•  Solution: continues flow > removal of inhibitory products and
provision of fresh “substrates”
•  first realized in cell-free translation systems with matriximmobilized template polynucleotides
Spirin, et al. Science. 1988 Nov 25;242(4882):1162-4.
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Translation in cell free extracts
• 
Components to be added
–  amino acids
–  ATP, GTP
–  energy-regenerating system
(PEP + pyruvate kinase)
–  RNase and protease inhibitor
• 
Commercially available
–  additives
–  e.g. chaperones
Rapid translation system (RTS,Roche)
•  E. coli extract
•  RTS ProteoMaster
– 
– 
– 
• 
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9.400 € !!!
Heating and cooling 20-50 °C
Shaking 120 - 990 rpm
Timer 00:01 - 99:59 hours
RTS500 high yield kit
>>up to 5 mg protein within 24 h
S-S bond formation
Folding problems
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Coupled transcription / translation
Plasmid
T7P
RBS
GENE OF INTEREST
ATG
TRANSCRIPTION
tag stop
T7T
T7 RNA POLYMERASE
mRNA
GENE OF INTEREST
TRANSLATION
O T E
P R
E. coli LYSATE
I N
I
N
S T
T
F
T A
G
Protein
E R E
O
Continuous Exchange Cell Free
(CECF)- Principle
Protein
DNA
reaction
chamber
inhibitory
by-products
membrane
supply
feeding
chamber
• DNA template
• T7 RNA polymerase
• Translation machinery E. coli
• Energy regenerating system
• Amino acids
• Energy substrates
• Buffer components
• NTPs
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Large volume CECF solutions
High trough put solutions
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Advantage of continuous exchange
The PURE System
“protein synthesis using recombinant elements”
Nat Biotechnol. 2001 Aug;19(8):732-3.
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E. coli extract
Lot dependent
reproducibility ?
error rate ?
costs for 10 ml extract 6.000-8.000 €
Rabbit retikulocyte lysate
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Reticulocytes
•  the last stage before the mature red
blood cells are formed
•  darkly staining vesicles of
reticulocytes are fragments of former
endoplasmic reticulum
•  under normal conditions less than 1 %
of the circulating red blood cells are
reticulocytes
•  reticulocytes are specialized for
translation of globin
Rabbit retikulocyte lysate
• 
unique among eukaryotic cell free translation systems
• 
translates with same rate as cell for the first hour
• 
but requires removal of endogenous mRNA (globin) for low
background
• 
high content of hemoglobin (>90% protein content)
• 
rabbits are made anaemic by treatment with acetylphenylhydrazin
• 
about 40 ml of lysate from one rabbit
• 
less dependent on capping than wheat germ extract
• 
hemin treatment to suppress inhibition of eIF2alpha
• 
Jackson & Hunt, Meth. in Enzymology, Vol. 96, page 50 - 74
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Wheat germ lysate
•  source is cheap
•  vitamin-rich embryo of the wheat kernel that is
separated before milling
•  lysate gives higher yield than retic lysate
Wheat germ lysate
• 
does not require removal of endogenous mRNA
• 
more dependent on capping than reticulocyte lysate
• 
ionic optima for translation may vary for different templates
• 
tendency to produce incomplete translation products
• 
>>do not use for proteins larger than 60 kDa
• 
Creatine phosphate und creatine kinase as energy source
• 
recommended for mRNAs which contain small doublestranded base pairing regions
• 
Erickson & Blobel, Methods in Enzymology, 1983, Vol. 96, page 39-50
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Tritin is expressed in the endosperm and
depurinates endogenous 28S rRNA
thereby inhibiting ribosomes
embryo
washed
PNAS Vol. 97, Issue 2, 559-564, January 18, 2000
DHFR
washed embryos
conventional system
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In vitro translation using yeast
•  source is cheap
•  easy to cultivate in the lab
•  Fungi have cell wall containing
chitin and cellulose
•  enzymatic digestion prior to
cell disruption
•  depends on capping
•  advantage: extracts can be
made from mutant strains
S. cerevisiae
(at 1000 fold magnification)
Garcia PD, Hansen W, Walter; Methods Enzymol. 1991;194:675-82.
Hemin Structure
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Figure 1. Using special stains such as methylene blue or brilliant cresyl blue, reticulocytes
stain with dark blue granules whereas mature erythrocytes evenly stain pale blue
Maslak, P. ASH Image Bank 2005;2005:101299
Copyright ©2005 American Society of Hematology. Copyright restrictions may apply.
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