Transcript Technologie de l’ADN Recombinant CHMI 4226 F

Recombinant DNA Technology
CHMI 4226 E
Week of March 14, 2005
Expression of proteins into living
organisms
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Expression of cDNAs
cDNA
Introduction into organism
(bacteria, yeast, insects, plants, mammals)
Purification
Phenotype
Activity
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Localisation
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Why express recombinant
proteins?
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Expression into living organisms
Source: Genetic Engineering News. 2004, 24(18): 22-28.
Bacteria
•
•
Advantages:
–
–
–
–
•
Yeast
– Eukaryote – allows for posttranslational modifications
– Rapid growth
– High yield
– Inexpensive to scale up
– Secretes products into the
medium easily
– Splices pre-mRNA
Well understood
Inexpensive to scale up
Rapid growth
Wide choice of vectors and
modified strains
Disadvantages:
– No post-translational modifications
(glycosylation),which can affect
protein folding;
– Protein aggregation and formation
of inclusion bodies
– Purified protein may be
contaminated with E. coli-derived
products.
Advantages:
•
Disadvantages:
– Proteases may degrade the
foreign protein
– Hyperglycosylation
– Protein may aggregate or fold
improperly
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Expression into living organism
Source: Genetic Engineering News. 2004, 24(18): 22-28.
Insect cells
Mammalian cells
• Advantages:
• Advantages:
– High yield
– Proper post-translational
modifications
– Proteins secreted into medium
– Ability to express multiple genes
simultaneously
–
Proper post-translational
modifications
– Pre-mRNA properly spliced
• Disadvantages:
–
• Disadvantages:
– Slow growth
– More expensive than E. coli /yeast
– Requires expertise in insect cell
culture and baculoviral vectors
Slow growth
– Requires expertise in mammalian
cell culture
– Expensive
– Purified proteins may have viral
contaminants
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Expression in E. coli
• Procedure is relatively
straightforward;
• Cloning in the pGEX
vector is facilitated by
carefully designing PCR
primers before
amplification of the cDNA
encoding the protein of
interest
– ORF of protein must be in
frame with GST
– Appropriate restriction sites
for cloning.
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Expression in E. coli
• Frequently used to express high levels of a
protein which will then be purified;
• Protein is often expressed as a fusion with
glutathione S-transferase (GST) to facilitate the
purification procedure.
• A protease cleavage site is introduced between
GST and the protein of interest in order to
remove GST following purification procedure.
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pGEX vector
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Expression in E. coli
Cloning in pGEX
Added restriction sites
Protease cleavage site
ATG
STOP
GST
YFcDNA
Eliminate STOP codon
ORF of YFcDNA in frame
with GST
OR
ATG
STOP
ATG
GST
YFcDNA
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Expression in E. coli
• Cells are first lysed
• Protein extract is subjected to
affinity chromatography on
GSH-sepharose: GST (GluCys-Gly) binds GSH and is
retained on the column;
Glutathione sepharose bead
• The protein is then eluted as
follows:
– Addition of an excess of GSH
to the column;
– OR addition of protease to
cleave the protein of interest
from GST.
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Lane 1: Size
marker
Lane 2: Total E.
coli extract
Lane 3: Same
extract after
purification on
GST-sepharose
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Expression in E. coli
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Expression into living organisms
Source: Genetic Engineering News. 2004, 24(18): 22-28.
Bacteria
•
•
Advantages:
–
–
–
–
•
Yeast
– Eukaryote – allows for posttranslational modifications
– Rapid growth
– High yield
– Inexpensive to scale up
– Secretes products into the
medium easily
– Splices pre-mRNA
Well understood
Inexpensive to scale up
Rapid growth
Wide choice of vectors and
modified strains
Disadvantages:
– No post-trasnlational modifications
(glycosylation),which can affect
protein folding;
– Protein aggregation and formation
of inclusion bodies
– Purified protein may be
contaminated with E. coli-derived
products.
Advantages:
•
Disadvantages:
– Proteases may degrade the
foreign protein
– Hyperglycosylation
– Protein may aggregate or fold
improperly
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Expression in yeast – use of
Saccaromyces cerevisiae
• 2u origin: yeast replication
origin
• URA 3: selection of yeast
auxotrophic for uracil (allows
growth on uracil-deficient
media)
• Cyc1 TT: transcription
termination sequence of Cyc1
mRNA
• pGal promoter: induction of
expression when yeast are
grown in galactose-containing,
glucose-deficient media;
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Expression in yeast – use of Pichia
pastoris
• P. pastoris:
– Methylotrophic yeast: uses
methanol as sole carbon
source, yielding formaldehyde
and hydrogen peroxide (done
in peroxysomes);
– Protein glycosylation is closer
to mammalian cells;
– A mich higher biomass (10
times!!) can be obtained with
P. pastoris than S. cerevisiae,
yielding greater protein
amounts (10 to 100 fold!).
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Expression in yeast – use of Pichia
pastoris
•
Alcohol oxidase (AOX1) promoter:
–
–
–
•
Zeocin:
–
–
•
Amino acid sequence from c-myc protein
Used for easy detection by Western blot
(reviewd later…patience!)
a-factor:
–
–
•
an antibiotic
allows for selection of yeast containing
the vector
C-myc epitope:
–
–
•
Induced by methanol
Allows for high levels of foreign protein
expression (30% of all proteins in
methonal-induced P. pastoris is Alcohol
oxidase!!)
Repressed by glucose
Mating factor secreted by yeast cells;
Allows for secretino of the foreign protein
into the culture media;
6 x HIS:
–
–
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6 histidine residues added after the
protein of interest
Allow for easy purification by affinity
chromatography on a nickel column.
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Protein purification with 6 x His tag
•
Protein extracts are loaded on a
column with beads onto which Ni+2 is
bound;
•
The His residues of the tag will bind
the Ni+2, retaining the protein on the
column;
•
Elution is done by adding an excess
of imidazole to the column (imidazole
is the building block of the His side
chain).
•
Advantages:
– Smaller in size than GST
• less immunologically active
• Less interference with protein folding
– No need to remove from protein after
purification
– The interaction His-resin does not
depend on tag structure (unfolded
proteins can be purified).
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Expression into living organism
Source: Genetic Engineering News. 2004, 24(18): 22-28.
Insect cells
Mammalian cells
• Advantages:
• Advantages:
– High yield
– Proper post-translational
modifications
– Proteins secreted into medium
– Ability to express multiple genes
simultaneously
• Disadvantages:
– Slow growth
– More expensive than E. coli /yeast
– Requires expertise in insect cell
culture and baculoviral vectors
–
Proper post-translational
modifications
– Pre-mRNA properly spliced
• Disadvantages:
– Slow growth
– Requires expertise in mammalian
cell culture
– Expensive
– Purified proteins may have viral
contaminants
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Using insect cells for protein expression –
the baculovirus system
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Using insect cells for protein expression –
the baculovirus system
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Using insect cells for protein expression –
the baculovirus system
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Using insect cells for protein expression –
the baculovirus system
• Requires homologous recombination to create a
recombinant baculovirus.
• This recombination takes place between:
– a linearized version of the baculovirus genome with part of an
essential gene missing,
– and a transfer vector carrying the needed missing piece and the
desired gene.
• The recombinant retrovirus is then used to infect insect
cells, which will produce large amounts of the protein of
interest (which is under the control of the strong
polyhedrin promoter that normally controls formation of
the major baculovirus protein.).
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Using insect cells for protein expression –
the baculovirus system
Transfer vector
Cloned gene
5’
3’
x
x
Cloned gene
5’
3’
Polyhedrin gene
Recombinant
AcMNPV DNA
AcMNPV DNA
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Using insect cells for protein expression –
the baculovirus system
• Pph: polyhedrin promoter
for high expression
levels;
• TnTr/TnTL: sequences
used for the homologous
recombination between
the transfer vector and
the baculovirus genome;
• Gentamicin: gene for
resistance to the
antibiotic gentamicin;
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Using insect cells for protein expression
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Recombinant proteins produced with the
baculovirus system
a/b-interferon
Adenosine deaminase
Rhodopsine
CFTR
Erythropoietin
HIV envelope protein
Influenza hemagglutinin
protein
• Interleukin 2
•
•
•
•
•
•
•
• Malaria parasite proteins
• Mouse monoclonal
antibodies
• Poliovirus proteins
• Rabies virus glycoprotein
• Simian rotavirus capsid
antigen
• Tissue plasminogen
activator
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Expression into living organism
Source: Genetic Engineering News. 2004, 24(18): 22-28.
Insect cells
Mammalian cells
• Advantages:
• Advantages:
– High yield
– Proper post-translational
modifications
– Proteins secreted into medium
– Ability to express multiple genes
simultaneously
–
Proper post-translational
modifications
– Pre-mRNA properly spliced
• Disadvantages:
–
• Disadvantages:
– Slow growth
– More expensive than E. coli /yeast
– Requires expertise in insect cell
culture and baculoviral vectors
Slow growth
– Requires expertise in mammalian
cell culture
– Expensive
– Purified proteins may have viral
contaminants
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Protein expression in
mammalian cells
• 1. Clone cDNA of interest
• 2. Introduce modifications
(mutations):
– Increase stability (e.g. in
blood)
– Improve enzymatic activity
– Decrease antigenicity
• 3. Introduce into
mammalian cells
(transfection)
• 4. Purification
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Protein expression in mammalian
cells– tissue plasminogen activator (tPA)
Modifications
Stability in
plasma
Fibrin
binding
Activity
against
clots
Thr(103)Asn
10
0.34
0.56
Lys-His-Arg-Arg (296-299)
Ala-Ala-Ala-Ala
0.85
0.93
1.01
Thr(103)Asn
+
Asn (117)Gln
3.4
1
1.17
0.87
0.85
Thr(103)Asn + Asn (117)Gln
8.3
+
Lys-His-Arg-Arg (296-299)
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Ala-Ala-Ala-Ala
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Mammalian expression vector
Promoter
Polyadenylation signal
Antibiotic resistance gene
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Mammalian Promoters
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Useful Mammalian Promoters
• Viral promoters
• Cellular promoters:
– Constitutive:
– Constitutive:
• CMV
• SV40
• Ubiquitin (UbC)
• Thymidine kinase
• Human eIF1a
– Inducible:
• MMTV (glucocorticoids)
– Inducible:
• Heat shock (42oC)
• Metallothioneine (zinc)
– Restricted expression:
• Lck: thymocytes
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Antibiotic resistance genes
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Optimization of translation in
mammalian cells
• Kozak consensus sequence:
(GCC)GCCA/GCCATGG
• Ensures optimal translation initiation;
• Can easily be added by PCR-mediated
mutagenesis.
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Epitope tagging
• Epitope tags: facilitate the detection of the protein of
interest with «generic» antibodies.
• Tag added either at the N- or C-terminal of the ORF (in
that latter case: BEFORE the stop codon!!)
• Tag can easily be added through PCR-mediated
mutagenesis.
HA-tag: Tyr-Pro-Tyr-Asp-Val-Pro-Asp-Tyr-Ala
HIS-tag: His-His-His-His-His-His
Myc-tag: Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu
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Mammalian cell transfection
Stable vs transient
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Transfection procedures
Calcium Phosphate
precipitation
Electroporation
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Lipofection
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Cell transduction
• Viruses are widely used to introduce DNA
molecules into mammalian cells;
• Form the basic vehicle for gene therapy
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Cell transduction
Retroviruses
http://www.clontech.com/upload/images/tools/retroviral/RetroDiagram.gif
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http://home.ncifcrf.gov/hivdrp/RCAS/images/figure1_870x660.gif
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Cell transduction
Retroviral vectors
•
Advantages:
– 100% transduction can be
acheived
– Integrates in the genome: stable
tranduction is possible
•
Disadvantages:
– Limited to actively proliferating
cells (so: neurons cannot be
transduced with retroviruses);
– Low titers (106-107)
http://www.clontech.com/upload/images/tools/retro/images/retro7.jpg
– Integration is random and can
lead to insertional mutagenesis:
insertion in the genome may lead
to cancer.
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Cell transduction
Retroviruses
http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=rv.section.4357
http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=rv.section.4357
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Cell transduction
http://catalog.takara-bio.co.jp/en/IMAGES/6160b_en.gif
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http://www.nature.com/gt/journal/v12/n14/images/3302570f1.jpg
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http://www.biotechnology.uwc.ac.za/StaffandStudents/Staff/Sean/Virology%20Lecture%20Gene%20Therapy_files/image008.jpg
Retrovirus production
Cell transduction
• Adenovirus:
Adenoviruses
– DNA virus
– Endemic in humans
– Infects human cells by biding to cell
adhesion receptors common to most
cells
• Adenoviral vectors:
– Advantages:
• Infects wide variety of cells (replicating
and non-replicating)
• High titers are acheived (1012)
• Does not integrate in genome: no
insertional mutagenesis
– Disadvantages:
• Does not integrate in genome
(application is limited to transient
transfection)
• Causes immune response in humans
(limits in vivo applications) CHMI 4226E - W2009
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Cell transduction
Adenoviruses
http://dels.nas.edu/ilar_n/ilarjournal/45_3/graphics/45_3_337f2.jpg
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http://www.qbiogene.com/products/adenovirus/images/figure1-large.gif
Cell transduction
Adenoviruses
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Transfection procedures
• The choice of the transfection procedure
depends on several considerations:
– The type of molecule transfected
(oligonucleotide, RNA, DNA);
– The cell line (suspension, adherent);
– The downstream application (importance of
high vs low transfection efficiency).
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Fusion protein
Green fluorescent Protein (GFP)
• GFP:
– Protein isolated
from the jelly fish
– Glows green on its
own when exposed
to UV light!
– Very useful to
« see » your
favorite protein!
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Fusion protein
Green fluorescent Protein (GFP)
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Fusion protein
Green fluorescent Protein (GFP)
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Fusion protein
Green fluorescent Protein (GFP)
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Fusion protein
Green fluorescent Protein (GFP)
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Fusion protein
Green fluorescent Protein (GFP))
Golgi
apparatus
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Fusion protein
Green fluorescent Protein (GFP)
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