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Cell-free expression based
protein microarrays
Cell-free expression involves the rapid, in situ
synthesis of proteins from their corresponding DNA
templates directly on the microarray surface. Protein
arrays generated by this technique have shown great
potential in eliminating the drawbacks of traditional cellbased methods.
Harini Chandra
Affiliations
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Master Layout (Part 1)
This animation consists of 5 parts:
Part 1 – Protein in situ array (PISA)
Part 2 – Nucleic Acid Programmabe Protein Array (NAPPA)
Part 3 – Multiple Spotting Technique (MIST)
Part 4 – DNA Array to Protein Array (DAPA)
mRNA
Part 5 – HaloTag technique
Cell-free
lysate
Transcription
Ribosomes
Translation
3
PCR generated DNA
construct
4
5
Tagged
protein
Tag-capturing
agent
He, M., Taussig, M. J., Single step generation of protein arrays from DNA by cell-free expression and in situ
immobilisation (PISA method). Nucleic Acids Res. 2001, 29, e73.
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Definitions of the components:
Part 1 – Protein in situ array (PISA)
1. PCR generated DNA construct: The DNA construct used for protein
expression is generated by PCR and encodes the protein of interest, a T7
promoter, sequences for initiation of translation, suitable termination
sequences as well as an N- or C-terminal tag sequence for immobilization
onto the microarray surface.
2. Cell-free lysate: Crude cell lysates obtained from growing cells contain
all the essential machinery that are required for transcription and translation
to take place. Nucleotides, essential amino acids and other energygenerating factors are added exogenously. Commonly used expression
systems include E. coli S30, wheat germ extract (WGE) and rabbit
reticulocyte lysate (RRL).
3. Transcription: The process by which mRNA is synthesized from its
corresponding DNA template with the help of suitable enzymes and factors.
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4. mRNA: The messenger RNA (mRNA) is transcribed from a DNA
template and contains the coding information for the corresponding protein
product.
5. Ribosomes: These are the site of synthesis of proteins from their
respective mRNA. They are made up of a large and small subunit and carry
out translation with the help of tRNAs, enzymes and other elongation and
termination factors.
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Definitions of the components:
Part 1 – Protein in situ array (PISA)
6. Translation: The process by which the mRNA code is converted into its
protein sequence. The mRNA is read in the form of three letter codes
known as codons which specify one amino acid.
7. Tagged protein: The protein synthesized by translation has a specific
tag molecule that allows the protein to get immobilized onto the array
surface as soon as it is produced. (eg. His6 tag).
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4
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8. Tag-capturing agent: This is a specific molecule that has been coated
onto the array surface so that the protein obtained by cell-free synthesis
will get captured onto the array due to the specific binding interaction
between the tag and tag-capturing agent. (eg. Nickel-nitrilo triacetic acid,
Ni-NTA).
Part
1,
Step
1:
1
Tag-capturing
agent
2
3
4
5
Array surface
Protein microarray
Action
Description of the action
One of the spots First show the parallelogram with dots
on the protein
as shown. Zoom into one of the dots
microarray must and show the figure drawn above.
be zoomed into
and the rest must
be shown.
Audio Narration
In PISA, the protein microarray surface is
coated with a suitable tag-capturing agent that
can immobilize the protein of interest through
specific interactions once it is produced.
Part
1,
Step
2:
1
Transcription
mRNA
Ribosomes
2
PCR generated DNA
construct
3
Translation
Tagged
protein
RNA Polymerase
(part of cell-free
lysate)
Tag-capturing
agent
4
5
Action
Description of the action
Audio Narration
As shown First show the yellow strands followed by the green The protein is expressed from its corresponding
oval moving along them and then the appearance of DNA using cell-free lysates such as E.coli S30 or
in the
animation the green strands. Next show the blue round
rabbit reticulocyte lysate (RRL). The tagged protein
structures moving along the green strands and
is then captured specifically onto the array surface
appearance of the blue protein shapes. This is
followed by the surface at the bottom with the moon through the tag-capturing agent. PISA successfully
shape. The oval must move down and get attached to overcame drawbacks of cell-based techniques
this as shown.
such as protein insolubility, aggregation etc.
Master Layout (Part 2)
1
This animation consists of 5 parts:
Part 1 – Protein in situ array (PISA)
Part 2 – Nucleic Acid Programmable Protein Array (NAPPA)
Part 3 – Multiple Spotting Technique (MIST)
Part 4 – DNA Array to Protein Array (DAPA)
Part 5 – HaloTag technique
2
Translation
Transcription
3
GST tag
Cell-free
lysate
cDNA +GST
GST
tag
GST
GST
Anti-GST
antibodies
4
BSA
5
Aminosilane coated glass slide
BS3
Ramachandran, N., Raphael, J. V., Hainsworth, E., Demirkan,G. et al., Next-generation high-density self-assembling functional
protein arrays. Nat. Methods 2008, 5, 535–538.
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2
3
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5
Definitions of the components:
Part 2 – Nucleic acid programmable protein array (NAPPA)
1. Aminosilane coated glass slide: A glass slide coated with aminosilane
reagent is used for cell-free protein expression in NAPPA.
2. cDNA+GST tag: High quality cDNA containing the gene to be expressed
as its glutathione-S-transferase (GST) fusion. This is immobilized onto the
array surface.
3. BSA: The protein bovine serum albumin (BSA) is added as part of the
master-mix. This improves the binding efficiency of the cDNA onto the array
surface.
4. BS3: BS3 is a cross-linking agent that facilitates immobilization of the
capture antibody on to the array surface. BS3 is also added as part of the
NAPPA master-mix.
5. GST tag: The protein obtained from translation of the mRNA is generated
as a GST fusion i.e. it has a GST tag that can specifically bind onto the
microarray surface through the antibody immobilized on the surface.
6. Anti-GST antibodies: Antibodies that bind specifically to the GST tag on
the synthesized protein are immobilized on the surface of the array thereby
allowing the expressed protein to be co-localized with its corresponding
DNA. This technique therefore does not produce pure protein arrays but is
capable of generating very high density microarrays.
Part
2,
Step
1:
1
GST GST
GST
cDNA +
GST tag
Anti-GST
antibody
BSA
2
BS3
Aminosilane
coated glass slide
3
4
5
NAPPA master mix
Protein microarray
Action
One of the spots
on the protein
microarray must
be zoomed into
and the figure on
topmust be
shown.
Description of the action
First show the parallelogram with
dots as shown. Next the hand must
move across the surface and then
disappear with gradual appearance
of the pink solution on top. Next,
zoom into one of the dots and show
the figure drawn above.
Audio Narration
An aminosilane-coated glass slide forms the array
surface for NAPPA. To this, the NAPPA master mix is
added which consists of BSA, BS3, GST-tagged cDNA
and the anti-GST capture antibodies. The BSA
improves efficiency of immobilization of the cDNA onto
the array surface while the BS3 cross-linker facilitates
binding of the capture antibody.
Part
2,
Step
2:
1
Ribosomes
mRNA
Translation
Transcription
2
3
GST tag
cDNA + GST
GST
tag
GST
GST
RNA Polymerase
(part of cell-free
lysate)
Anti-GST
antibodies
BSA
4
BS3
Action
5
As
shown
in the
animatio
n.
Description of the action
First show the surface at the bottom bound to the green Y
shaped objects and yellow strands. Then the pink circles
must appear and move up as shown followed by the arrow
and the purple strands. The green shape must appear on
top of the purple strand and move across the strand as
shown. The yellow shape with the triangle then appears
and this must move in such a way that it binds to the green
shapes below.
Audio Narration
The cDNA is expressed using a cell-free extract
to give the corresponding protein with its GST tag
fused to it. This tag enables capture of the protein
onto the slide by means of anti-GST antibodies.
NAPPA technique can generate very high density
arrays but the protein remains co-localized with
the cDNA.
Master Layout (Part 3)
1
This animation consists of 5 parts:
Part 1 – Protein in situ array (PISA)
Part 2 – Nucleic Acid Programmabe Protein Array (NAPPA)
Part 3 – Multiple Spotting Technique (MIST)
Part 4 – DNA Array to Protein Array (DAPA)
Part 5 – HaloTag technique
2
Tagged
detection
antibody
Second
spotting
3
Expressed
protein
First spotting
4
5
Cell-free
lysate
DNA
template
Angenendt, P., Kreutzberger, J., Glokler, J., Hoheisel, J. D., Generation of high density protein microarrays
by cell-free in situ expression of unpurified PCR products. Mol. Cell.Proteomics 2006, 5, 1658–1666.
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2
Definitions of the components:
Part 3 – Multiple spotting technique (MIST)
1. First spotting: This step involves the spotting of DNA template coding
for the protein of interest in as little as fg quantities, onto the solid array
surface.
2. DNA template: The DNA that codes for the protein of interest and is
expressed by transcription and translation.
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3. Second spotting: After the template DNA is spotted on to the array
surface, the cell-free lysate is then transferred exactly on top of the first
spot. This second spotting step marks the beginning of expression of the
template DNA.
4. Expressed protein: The proteins produced by cell-free expression
from the corresponding DNA templates are immobilized on the array
surface either through a tag-capturing agent or more commonly, by
means of non-specific interactions. These proteins can then be detected
by suitably tagged antibodies.
5. Tagged detection antibody: Antibodies specific to the protein of
interest can be added to the array surface for detection. The fluorescent
tag molecules will indicate protein expression upon specific binding
interactions between the protein and antibody.
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Part
3,
Step
1:
1
DNA
template
2
First spotting
3
Protein microarray
4
Action
5
As
shown
in the
animatio
n.
Description of the action
First show the parallelogram with dots. Then show the
hand moving down into one of the spots followed by
appearance of text ‘first spotting’ and then disappearance
of hand and text.
That spot must then be zoomed into and the figure on top
must be shown.
Audio Narration
The first spotting step of the multiple spotting
technique, which is also capable of producing
high density arrays, involves the addition of
template DNA on to the solid array support.
The template DNA can even be in the form of
unpurified PCR product, one of the major
advantages of this technique.
Part
3,
Step
2:
1
Cell-free
lysate
DNA
template
2
Second
spotting
3
Protein microarray
4
Action
5
As
shown
in the
animatio
n.
Description of the action
First show the parallelogram with dots. Then show the
hand moving down into one of the spots followed by
appearance of text ‘second spotting’ and then
disappearance of hand and text.
That spot must then be zoomed into and the figure on top
must be shown.
Audio Narration
The second spotting step involves the
addition of the cell-free lysate directly on top
of the first spot. Transcription and translation
can begin only after the second spotting step.
Part
3,
Step
3:
1
Ribosomes
mRNA
Tagged
detection
antibody
2
Expressed
protein
3
Cell-free
lysate
RNA
Polymerase
4
Action
5
As
shown
in the
animatio
n.
Description of the action
First show the grey surface with the green strands and
the grey cloud on top. Then show the pink circles along
with their label . These circles must move up as shown
in the animation followed by appearance of the arrow.
The pale purple ‘protein’ must then appear followed by
the figure labelled as ‘tagged antibody’. This figure
must move diagonally as shown until it binds to the pink
shape.
Audio Narration
The protein expressed from the template DNA
binds to the array surface by means of nonspecific interactions, one of the drawbacks of
this procedure. A detection antibody specific to
the protein of interest is then added which
indicates protein expression levels by means of
a suitable fluorophore.
Master Layout (Part 4)
1
2
This animation consists of 5 parts:
Part 1 – Protein in situ array (PISA)
Part 2 – Nucleic Acid Programmabe Protein Array (NAPPA)
Part 3 – Multiple Spotting Technique (MIST)
Part 4 – DNA Array to Protein Array (DAPA)
Part 5 – HaloTag technique
Ni-NTA coated slide
DNA template
Lysatecontaining
permeable
membrane
3
4
Tagged,
expressed
protein
Protein tag-capturing
agent
Ni-NTA coated slide
5
He, M., Stoevesandt, O., Palmer, E. A., Khan, F. et al., Printing protein arrays from DNA arrays. Nat. Methods
2008, 5, 175–177.
1
Definitions of the components:
Part 4 – DNA array to protein array (DAPA)
1. Ni-NTA coated slide: The microarray slide surface is coated with
Nickel-nitrilotriacetic acid (Ni-NTA) which acts as a useful capture agent.
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3
4
2. DNA template: PCR amplified DNA that codes for the protein of
interest is immobilized on a Ni-NTA coated slide. This slide can be reused
several times for generation of protein.
3. Lysate containing permeable membrane: A permeable membrane
that is soaked with the cell-free extract is placed in between the
immobilized DNA template slide and a slide having the protein tagcapturing agent. The newly expressed proteins penetrate the membrane
and bind to the protein purification slide.
4. Tagged, expressed protein: The newly expressed protein bearing the
tag molecule that is formed in the permeable membrane, slowly
penetrates the membrane and gets immobilized by means of its tagcapturing agent.
5. Protein tag-capturing agent: A molecule that will specifically bind to
the protein of interest thereby immobilizing it onto the Ni-NTA coated slide.
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Part
4,
Step
1:
1
Ni-NTA coated slide
DNA template
2
3
Lysatecontaining
permeable
membrane
Tagged,
expressed
protein
Protein tag-capturing
agent
Ni-NTA coated slide
4
Action Description of the action
5
As
shown
in the
animatio
n.
First show the slide on top and the slide below along
with the grey oval in between. Then show the dark
green circles on the red strands which must move
downwards as shown. Then show appearance of
the maroon strands and then the green figures on
top of it. This must move from left to right as shown.
Then show appearance of the light blue figure
(protein) which must slowly move down until it gets
captured by the dark blue pie-shaped circles below.
Audio Narration
The slides bearing the DNA template and the protein
tag-capturing agent are assembled face-to-face with a
lysate containing permeable membrane placed in
between. The expressed protein slowly penetrates the
membrane and gets immobilized on the slide surface
through its capture agent. The DNA template array
can be reused several times in this method.
Master Layout (Part 5)
1
2
This animation consists of 5 parts:
Part 1 – Protein in situ array (PISA)
Part 2 – Nucleic Acid Programmabe Protein Array (NAPPA)
Part 3 – Multiple Spotting Technique (MIST)
Part 4 – DNA Array to Protein Array (DAPA)
Part 5 – HaloTag technique
mRNA
Cell-free lysate
Transcription
3
Ribosomes
Translation
DNA construct
HaloTag
bound
protein
4
HaloTag
ligand
5
Nath, N., Hurst, R., Hook, B., Meisenheimer, P. et al., Improving protein array performance: Focus on
washing and storage conditions. J. Proteome Res. 2008, 7, 4475–4482.
1
2
3
Definitions of the components:
Part 5 – HaloTag technique
1. DNA construct: The template DNA coding for the protein of interest
along with the HaloTag.
2. HaloTag bound protein: HaloTag is a 33kD engineered derivative of
bacterial hydrolase that can be used to tag the desired protein. The mode
of interaction between HaloTag and its ligand is through covalent bonding,
thereby ensuring firm capture of the protein on the array surface without
any material loss during washing. It also prevents any loss of protein
function as it allows for oriented capture of the protein on to the array
surface.
3. HaloTag ligand: The PEG-coated glass slide is activated with the
HaloTag ligand for oriented and firm capture of the expressed protein on
to the array surface.
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5
Part
5,
Step
1:
1
HaloTag
ligand
2
3
4
5
Array surface
Protein microarray
Action
Description of the action
One of the spots First show the parallelogram with dots
on the protein
as shown. Zoom into one of the dots
microarray must and show the figure drawn above.
be zoomed into
and the rest must
be shown.
Audio Narration
The slide is activated with the HaloTag ligand
which captures the expressed protein through
firm covalent interactions thereby preventing
any material loss and ensuring oriented
capture of the protein.
Part
5,
Step
2:
1
Transcription
mRNA
Ribosomes
2
Translation
DNA construct
3
RNA Polymerase
(part of cell-free
lysate)
HaloTag
ligand
4
5
Action
Description of the action
HaloTag
bound
protein
Firm
covalent
capture
Audio Narration
As shown First show the purple strands followed by the The HaloTag fused protein is expressed using
green oval moving along them and then the
in the
lysates like RRL or WGE and covalently captured
animation appearance of the blue strands. Next show the on to the array surface through the HaloTag ligand.
pink round structures moving along the blue
strands and appearance of the yellow protein The specific interaction ensures oriented capture of
shapes. The green shape on the yellow protein the protein thereby preventing any possible
must then move down and get attached to the functional loss.
blue tags at the bottom as shown.
1 Interactivity option 1:Step No:1
Arrange the following components of cell-free expression in the right order for
protein synthesis to occur.
2
C) Tagged array
surface
A) Transcription
initiation factors
B) Translation
factors
E) Template DNA
encoding tag sequence
D)Amino acids
F) Cell-free lysate
containing enzymes &
ribosomes
3
1
2
3
4
5
6
4
Interacativity Type
Drag and drop
5
Options
User must be
allowed to drag
and drop the
images given
above into the
numbered
circles below.
Boundary/limits
Results
The correct order is E, F, A, B, D,
C. If the user gets this right, there
must be a pop-up which says
‘Correct answer’ else a popup
saying ‘incorrect, try again’. User
can try until he gets the correct
answer.
1
Questionnaire
1. Which of the following techniques allows the DNA template slide to be reused several
times?
2
Answers: a) NAPPA b) MIST c) DAPA d) HaloTag
2. Which technique ensures oriented capture of the expressed protein?
Answers: a) PISA b) HaloTag
3
c) NAPPA
d) MIST
3. Traditional cell-based techniques for microarray generation suffer from which of the following
drawbacks?
Answers: a) Protein expression in heterologous systems b) Protein purification c) Maintaining
protein stability d) All of the above
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5
4. What is one of the major drawbacks of the NAPPA technique?
Answers: a) It cannot generate high density arrays b) The immobilized DNA arrays cannot be
stored for long c) It requires large volumes of cell-free lysate d) Cloning procedures need
to be carried out to generate cDNA.
5. Which of the following techniques can make use of unpurified PCR generated DNA product?
Answers: a) PISA b) NAPPA c) DAPA d) MIST
Links for further reading
Books:
New and Emerging Proteomic Techniques. Edited by Dobrin Nedelkov & Randall W.Nelson
(Humana Press).
Research papers:





Chandra, H. & Srivastava, S. Cell-free synthesis-based protein microarrays and their applications.
Proteomics 2010, 10, 1-14.
He, M., Stoevesandt, O., Taussig, M. J., In situ synthesis of protein arrays. Curr. Opin. Biotechnol. 2008,
19, 4–9.
Jackson, A. M., Boutell, J., Cooley, N., He, M., Review: cell-free protein synthesis for proteomics. Brief
Funct. Genom.Proteomic 2004, 2, 308–319.
He, M., Taussig, M. J., Single step generation of protein arrays from DNA by cell-free expression and in
situ immobilisation (PISA method). Nucleic Acids Res. 2001, 29, e73.
Ramachandran, N., Hainsworth, E., Bhullar, B., Eisenstein,S. et al., Self-assembling protein mircoarrays.
Science 2004,305, 86–90.
Links for further reading
Research papers:




Ramachandran, N., Raphael, J. V., Hainsworth, E., Demirkan,G. et al., Next-generation high-density selfassembling functional protein arrays. Nat. Methods 2008, 5, 535–538.
Angenendt, P., Kreutzberger, J., Glokler, J., Hoheisel, J. D., Generation of high density protein
microarrays by cell-free in situ expression of unpurified PCR products. Mol. Cell.Proteomics 2006, 5,
1658–1666.
He, M., Stoevesandt, O., Palmer, E. A., Khan, F. et al., Printing protein arrays from DNA arrays. Nat.
Methods 2008, 5, 175–177.
Nath, N., Hurst, R., Hook, B., Meisenheimer, P. et al., Improving protein array performance: Focus on
washing and storage conditions. J. Proteome Res. 2008, 7, 4475–4482.