Transcript Cell-free expression systems

Cell-free expression systems
Cell-free systems make use of template DNA in the
form of plasmids or PCR products to carry out in vitro
transcription and translation. Protein microarray
generation using cell-free expression systems
eliminate the need for protein purification.
Harini Chandra
Affiliations
Master Layout (Part 1)
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This animation consists of 4 parts:
Part 1 – Commonly used cell-free systems
Part 2 – E. coli S30 extract
Part 3 – Wheat germ extract
Part 4 – In vitro protein synthesis
Cell-free expression systems
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3
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E. coli S30
Rabbit
reticulocyte
lysate (RRL)
Wheat germ
extract
(WGE)
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2
3
4
Definitions of the components:
Part 1 – Commonly used cell-free systems
1. Cell-free expression systems: Cell-free systems are used for in vitro
protein synthesis starting from DNA templates. These systems contain all
the necessary components and machinery for transcription and translation.
Some factors such as energy generating components, essential amino
acids etc. need to be added to the system for successful protein synthesis.
2. E. coli S30: This is a commonly used bacterial expression system that
is capable of producing protein yields of around ~6 mg/mL (Jackson et al.,
2004). This system however is not capable of carrying out posttranslational modifications (PTMs) of proteins due to the absence of
required machinery for this process and very often produces incomplete
protein chains. DNA templates obtained from bacterial sources are
commonly used with this cell-free lysate.
3. Rabbit reticulocyte lysate (RRL): A mammalian cell-free system that
also gives protein yields of ~6 mg/mL (Jackson et al., 2004). This system
is more suitable for expression of full length eukaryotic proteins from plant
and animal sources that require proper folding and PTMs.
4. Wheat germ extract (WGE): This is a cell-free expression system that
is capable of producing full length proteins with correct folding and PTMs
from bacterial, plant or animal sources. Yields obtained in this system are
however slightly lower than the E. coli and RRL.
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Part
1,
Step
1:
1
Cell-free expression systems
2
E. coli S30
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Rabbit
reticulocyte
lysate (RRL)
Wheat germ
extract
(WGE)
4
Action
5
Each of the
headings must
appear
sequentially
followed by
highlighting of
only the first
two as shown.
Description of the action
Audio Narration
(Please redraw all figures.)
First show the main heading on top appearing followed As given in the previous slide. To be
shown when the user selects one of the
by each of the sub-headings appearing in sequence
along with their respective figures. The user should be titles.
allowed to click on any of the three to obtain the
details as explained in the previous slide. Finally, the
first two must be highlighted as shown.
Master Layout (Part 2)
1
This animation consists of 4 parts:
Part 1 – Commonly used cell-free systems
Part 2 – E. coli S30 extract
Part 3 – Wheat germ extract (WGE)
Part 4 – In vitro protein synthesis
Cell lysis &
centrifugation
2
Extraction of
cell-free
system
Nucleoid
3
Active E. coli cell
Enzymes
4
Transcription
5
Circular
DNA
mRNA
Translation
Ribosomes
Expressed proteins
1. Schwarz, D., Dotsch, V., Bernhard, F. Production of membrane proteins using cell-free expression systems.
Proteomics 2008, 8, 3933-3946. 2. Biochemistry by A.L.Lehninger, 3rd edition (ebook)
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Definitions of the components:
Part 2 – E. coli S30 extract
1. Active E. coli cell: E. coli is possibly the most studied prokaryotic cell
and is easily available. The active state of this bacterium or any other cell
implies that it is in the process of synthesizing its proteins required for
various cellular functions. In this state, the cell produces various enzymes
and other factors in its cytoplasm that are required for the processes of
transcription and translation.
2. Nucleoid: The genetic material of the cell is present in its nucleoid in the
form of a single, circular molecule of DNA. Other circular DNA segments
known as plasmids may also be present in the cytoplasm.
3. Circular DNA: The genetic material of the E. coli cell that codes for the
various proteins required for cellular functioning and is responsible for
transmission of characteristics from one generation to the next.
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4. Enzymes: Biochemical catalysts that increase the rate of specific
reactions by as much as million fold. The various enzymes involved in the
process of transcription and translation such as RNA polymerase, peptidyl
transferase, amino acyl tRNA synthetase etc. are present in the cell-free
systems.
5. Transcription: The process by which template DNA is converted into its
complimentary mRNA sequence, which acts as the adaptor molecule
between DNA and proteins.
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2
Definitions of the components:
Part 2 – E. coli S30 extract
6. mRNA: The single stranded messenger RNA molecule that acts as the
link between the base sequence of DNA and the amino acid sequence of
proteins.
7. Ribosomes: These are complex and intricate cellular machines that
efficiently read the base sequence of the mRNA as triplet codons and
incorporate the corresponding amino acid into the growing polypeptide
chain.
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8. Translation: The process by which the mRNA in converted into its
corresponding amino acid sequence.
9. Expressed proteins: The proteins that are synthesized by the cellular
transcription and translation machinery which are required for various
functions of the cell.
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10. Extraction of cell-free system: The actively growing and functioning
cells are lysed and then centrifuged at 30,000g to collect the supernatant
containing cell-free extract. This has all the necessary components for in
vitro protein synthesis.
Part
2,
Step
1:
1
Nucleoid
2
Active E. coli cell
Essential
amino acids
Enzymes
3
ATP
N
Transcription
N
ATP
4
Action
5
N
ATP
The cell
structure must
be zoomed
into and the
animation
below must be
shown to be
taking place.
Translation
N
N N
Circular
Nucleotides
DNA
mRNA
Ribosomes
ATP
ATP
Expressed proteins
Description of the action
Audio Narration
(Please redraw all figures.)
First show the cell structure with its labels. This must be
zoomed into and several of the figures shown in the box
below must be shown. The 3 colored circles must then
move in a circle around the multi-colored circle (DNA)
followed by appearance of the colored strands and the
brown shapes on top of them. These must move across
the strand as depicted and the colored figures on the
right must then appear.
Actively growing and replicating E. coli cells can be used
for extracting cell-free lysates. These cells that are in the
process of growth and division are constantly producing
proteins and other factors required for the various cellular
processes. Co-factors and enzymes such as RNA
Polymerase, peptidyl transferase etc are available in
significant quantities due to cellular processes of
transcription and translation taking place in the cell.
Part
2,
Step
2:
1
Isolation of cell-free extract
Centrifugation
2
Lysis buffer
3
Supernatant –
cell free extract
Pellet – lysed
cells
Active E. coli cells
4
5
Action
The tube must
be shown to
enter the
instrument
shown and then
removed.
Description of the action
(Please redraw all figures.)
First show the tube with the particles as depicted.
Then show the tube being filled with the blue
solution and then being placed in the instrument.
The lid must then be closed and shown closed for
a few seconds after which the lid must be opened
and the tube taken out to show the solution as
shown on the right.
Audio Narration
The cells are lysed with a suitable buffer and then
centrifuged at 30,000g to collect the supernatant
containing the extract. Lysate that is extracted from
such actively growing and dividing cells will contain
all required cellular machinery to carry out in vitro
protein synthesis and requires addition of essential
amino acids, nucleotides, salts and other energy
generating factors.
Master Layout (Part 3)
1
This animation consists of 4 parts:
Part 1 – Commonly used cell-free systems
Part 2 – E. coli S30 extract
Part 3 – Wheat germ extract (WGE)
Part 4 – In vitro protein synthesis
Wheat seeds
2
Processing
steps
Selection &
washing
Clean
wheat
embryo
Wheat embryo
3
Extraction,
fractionation
& purification
Bran
Endosperm
4
5
Germ/embryo
Wheat germ
extract
Takai, K., Sawasaki, T., Endo, Y. Practical cell-free protein synthesis system using purified wheat embryos.
Nat Protoc. 2010, 5 (2), 227-238.
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Definitions of the components:
Part 3 – Wheat germ extract (WGE)
1. Wheat seeds: Seeds from the wheat grain are used for extraction of
the desired eukaryotic cell-free lysate. The structural components of the
wheat seed include:
a) Bran: This is the protective outer coating of the seed that is rich in
fibres, trace minerals and B vitamins.
b) Endosperm: The endosperm is the tissue that is produced
surrounding the embryo around the time of fertilization and provides the
seed with nutrition in the form of carbohydrates and proteins. It is this
endosperm that is the source of nutrients in processed wheat foods like
bread.
c) Germ/embryo: The reproductive part of a cereal that grows into a
plant and is a rich, concentrated source of several essential nutrients like
folate, Vitamin E, trace metals etc is known as the germ or embryo. It is
this part that contains the necessary cellular machinery and is therefore
used for preparation of the extract.
5. Processing steps: The seeds are processed in order to reach the
embryo by grinding them mildly followed by sieving to remove the outer
seed-coat fragments. The embryos are finally separated by addition of
organic solvents like cyclohexane or carbon tetrachloride for a short while
which cause the embryos to float thereby allowing them to be separated
and dried.
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3
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Definitions of the components:
Part 3 – Wheat germ extract (WGE)
6. Selection & washing: The obtained embryos are then carefully
screened to select only the good quality embryos that do not contain the
outer endosperm coating. These selected embryos are washed
thoroughly in cold water until any outer coating that may be present is
removed. The endosperm coating contains inhibitors of protein synthesis
and therefore must be removed.
7. Clean wheat embryo: The embryo that is free of any outer endosperm
coating and is ready for further processing.
8. Extraction, fractionation & purification: Cleaned embryos are
grinded with the extraction buffer followed by centrifugation at 30,000g at
4oC to separate the various components. The supernatant containing the
lysate is removed and purified by chromatography to separate any
components of the extraction buffer that are present.
9. Wheat germ extract: The desired, pure wheat germ extract is obtained
for in vitro protein synthesis.
Part
3,
Step
1:
1
Wheat seeds
Larger outer
coat fragments
Sieving
Grinding
2
Smaller
particles
3
Bran
Endosperm
Germ/embryo
Separated
embryos
Flotation
4
Action Description of the action
As
shown in
animatio
n.
5
(Please redraw all figures.)
First show the brown seeds, which must be zoomed into to
depict the figure below. It must then be zoomed out and
shown to enter the grey grinding machine. A powdery mass
must come out of it and must be shown to pass through a
mesh as shown on the right top. The fine particles coming
out of this mesh must then be shown in a blue liquid with
small particles on top and larger particles settling down.
Audio Narration
One of the most commonly used eukaryotic cell-free
expression systems is obtained from the embryo of
the wheat seeds. The seeds are grinded and then
sieved to remove their outer coating fragments. The
embryos and other small particles are floated in an
organic solvent like cyclohexane. The floating
embryos are quickly removed and dried to avoid any
damage from the organic solvent.
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Part 3, Step 2:
Extraction buffer
2
3
4
5
Grinding &
extraction
Clean embryos
Selected embryos
washed
Action Description of the action
Particles
First show the particles on the left followed by the blue
shown on solution appearing over it. The blue solution must then be
left must be poured out and this procedure must be repeated three
washed
times followed by appearance of the grey particles in the
with water middle panel. This is followed by addition of these
and then
particles into the blue solution in the container, The
mixed with particles must be shown to break into several smaller
the solution fragments when this happens.
on the right.
Audio Narration
The dried embryos are then carefully sorted such
that only the good embryos without any
endosperm coating are selected. The endosperm
contains certain inhibitors of protein synthesis that
must be removed. The selected embryos are
washed thoroughly with cold water after which
they are mixed with the extraction buffer and
grinded.
1
Part 3, Step 3:
Centrifugation
2
Wheat germ
extract
3
4
Action Description of the action
As shown
in
animation.
5
(Please redraw all figures.)
First show the solution in the tube with the particles
at the bottom. This tube must then enter the
instrument shown and the lid must be closed. It must
then be opened after a couple of seconds and the
tube must be removed from the instrument to show
the solution as shown on the right.
Audio Narration
This solution must be centrifuged at 30,000g at 4oC
which results in the wheat germ extract forming a
layer in between the top fatty layer fraction and the
pellet at the bottom. This fraction can be separated
and then purified by chromatographic methods to
remove any components of the extraction buffer. This
cell free lysate is capable of synthesizing full length
eukaryotic proteins with yields of around 4 mg/mL.
Master Layout (Part 4)
1
This animation consists of 4 parts:
Part 1 – Commonly used cell-free systems
Part 2 – E. coli S30 extract
Part 3 – Wheat germ extract (WGE)
Part 4 – In vitro protein synthesis
DNA template
2
Promoter
sequence
3
Ribosome
binding
site
Exogenous
factors
Cell-free
lysate
Essential
amino acids
tRNA
4
Enzymes
Termination
sequence
Gene of
interest
ATP
Ribosomes
ATP
ATP
N
N
N
N
N
Nucleotides
5
N
1
2
Definitions of the components:
Part 4 – In vitro protein synthesis
1. DNA template: The sequence coding for the protein of interest which
must be transcribed and translated using the suitable expression system.
This DNA may either be in the form of PCR DNA or plasmid DNA.
2. Promoter sequence: The region of DNA located upstream of the gene
of interest and having a specific base sequence that facilitates
transcription by binding to the sigma subunit of RNA polymerase enzyme.
Commonly used promoters include T7, SP3 or T3.
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3. Ribosome binding site: For translation to take place from the
synthesized mRNA, a translation initiation site is required which will allow
the ribosome and other factors required for the process to bind to it.
Translation initiation sequences such as the Shine-Dalgarno for
prokaryotes and Kozak sequence for eukaryotes are added for in vitro
protein synthesis.
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4. Gene of interest: The gene that codes for the desired protein or
polypeptide target. This may be obtained through PCR amplification or
may be present in the form of a plasmid.
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5. Termination sequence: For transcription and translation to be
appropriately halted, termination sequences must be present so that the
desired protein is correctly synthesized.
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2
3
4
5
Definitions of the components:
Part 4 – In vitro protein synthesis
6. Cell-free lysate: As described in Part 1, the cell-free lysate contains all
the necessary cellular machinery for protein synthesis to take place. The
lysate is extracted from an actively growing cell and therefore contains
enzymes, ribosomes, tRNA molecules, non-essential amino acids etc.
Mammalian cell-free lysates like wheat germ extract (WGE) and rabbit
reticulocyte lysate (RRL) allow post translational modifications to occur in
the protein while bacterial lysates like E. coli extract do not possess the
required enzymes for this process.
7. Exogenous factors: In addition to the cell-free lysate, certain
exogenous factors like essential amino acids, nucleotides, energy
generating factors etc. must be added to the system for the process to
take place successfully.
Part
4,
Step
1:
1
DNA template stored on
ice which must have the
following components
2
3
Promoter
sequence
Ribosome
binding
site
Gene of
interest
Termination
sequence
4
Action Description of the action
5
Zoom into the
tube shown on
top and then
show each
region being
highlighted as
shown in the
animation.
(Please redraw all figures.)
First show the tube being placed in the
vessel on top with cubes of ice. The tube
must be zoomed into and then show the
DNA template in the grey scale and then
appearance of each region starting from
the left. As the label for each region
appears, that region must be highlighted
as shown in the animation.
Audio Narration
The DNA template is thawed and then placed on ice during the
preparatory process. For in vitro protein synthesis to take place,
the DNA template must contain the gene coding for the protein
of interest. In addition to this, there must be a promoter
sequence which can initiate the transcription process, a
translation initiation sequence for binding of the ribosome as
well as suitable termination sequences to correctly synthesize
only the protein of interest.
1
Part 4, Step 2:
Essential
amino acids
ATP
ATP
ATP
2
N
N
N
N
N
N
Nucleotides
3
4
DNA template
stored on ice
Exogenous
factors
tRNA
Enzymes
Action Description of the action
As shown
in
animation.
5
Cell-free
lysate
(Please redraw all figures.)
First show the tube stored in the vessel on ice followed by appearance of
the blue tube which must be zoomed into and the blue cloud must be
shown. It must be zoomed out and then hand must be shown to add
solution to the grey tube as depicted. Next the orange tube must appear.
Again, it must be zoomed into and the orange cloud must be shown
followed by zooming out and the hand motion adding solution to the grey
tube. Color of the solution in grey tube must change upon addition.
Ribosome
Audio Narration
The thawed cell-free lysate containing the
essential cellular machinery for protein
synthesis is added to the DNA template
followed by the other exogenous factors
that are required for the process. All these
are done while storing the template on
ice to ensure that there is no loss of
activity.
1
Part 4, Step 3:
30oC
2
Incubate at
30oC
N
DNA template
Enzymes
ATP
N
N
N
ATP
N
N
Cell-free lysate
3
N
N
N
N
N
Nucleotides
N
ATP
ATP
tRNA
mRNA
Essential
amino acids
ATP
Ribosomes
4
5
Action
As
shown
in
animat
ion.
Description of the action
(Please redraw all figures.)
First show the tube being placed in the box on top and then
zoom into the tube. In that, show ‘DNA template’ followed by
the cloud on top along with the colored circles and their
respective labels. Then show the green ‘ATP’ circles and the
other chemical structures. The colored circles must then
move as shown and must result in the ‘mRNA’ structure
appearing below followed by the cloud and all other
components shown. The brown shapes must then move as
depicted.
Audio Narration
The tube containing all the required components is
then incubated at 30oC. Enzymes for transcription
bind to the promoter sequences and in the presence
of other factors like ATP and nucleotides, they carry
out synthesis of the mRNA transcript. This mRNA is
then translated into the corresponding protein with
the help of ribosomes, tRNA, enzymes and other
factors required for the process.
Interactivity
option
1:Step
No:1
1
Full length
Full length
Plant, animal &
bacterial
Plant, animal &
bacterial
Bacterial
Incomplete
E. coli S30
2
3
Posttranslational
modifications
Synthesized
protein
Template
source
Possible
Interacativity Type
Drag and drop.
5
WGE
Protein yield
~6 mg/mL
Possible
4
RRL
Not
possible
Options
Each row of the table must
appear one at a time and
three options to fill that row
must appear along with it.
User must drag and drop
the options into the correct
cell.
~4 mg/mL
Boundary/limits
~6 mg/mL
Results
The correct answers are depicted in
the animation above. At the end of
every row, if the user has got it
correct, a tick mark must appear at the
side else a cross. Once the entire
table is done correctly, a message
saying ‘Great job’ must appear.
Jackson, A. M., Boutell, J., Cooley, N., He, M. Cell-free protein synthesis for proteomics. Brief. Funct. Genom. Proteom. 2004,
2(4), 308-319.
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Questionnaire
1. Which of the following components will not be present in the WGE lysate?
Answers: a) Ribosomes b) RNA Polymerase c) Tryptophan d) peptidyl transferase
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2. Catalytic inhibitors of protein synthesis are found in which component of wheat seeds?
Answers: a) Embryo b) Endosperm c) Bran d) Germ
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3. Which of the following systems would be preferred for producing high yields of a full length
eukaryotic protein?
Answers: a) E. coli S30 b) WGE c) Xenopus oocyte d) RRL
4. The function of the Shine Dalgarno sequence is
4
Answers: a) Binding of RNA Polymerase b) Binding of sigma subunit c) Binding of tRNA d)
Binding of ribosome
5. Which of the following is not a promoter sequence?
Answers: a) Kozak b) T7 c) SP3 d) T3
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Links for further reading
Reference websites:
http://www.promega.com/applications/prtn_exp/expression.htm
Research papers:




Schwarz, D., Dotsch, V., Bernhard, F. Production of membrane proteins using cell-free
expression systems. Proteomics 2008, 8, 3933-3946.
Mikami, S., Masutani, M., Sonenberg, N., Yokoyama, S., Imataka, H. An efficient mammalian
cell-free translation system supplemented with translation factors. Protein Expression &
Purification 2006, 46, 348-357.
Katzen, F., Chang, G., Kudlicki, W. The past, present and future of cell-free protein synthesis.
Trends Biotechnol. 2005, 23 (3), 150-156.
Jackson, A. M., Boutell, J., Cooley, N., He, M. Cell-free protein synthesis for proteomics. Brief.
Funct. Genom. Proteom. 2004, 2(4), 308-319.