Recombinant DNA Technology CHMI 4226 E

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Recombinant DNA TechnologyCHMI 4226 E

• Week of March 14, 2005
• Expression of proteins into living organisms

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Expression of cDNAs
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Why express recombinant proteins?
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Expression into living organismsSource Genetic
Engineering News. 2004, 24(18) 22-28.

• Bacteria
• Advantages
• 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.

• Yeast
• Advantages
• Eukaryote allows for post-translational
  modifications
• Rapid growth
• High yield
• Inexpensive to scale up
• Secretes products into the medium easily
• Splices pre-mRNA
• 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
• 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

• Mammalian cells
• Advantages
• 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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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
Eliminate STOP codon
ORF of YFcDNA in frame with GST
OR
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Expression in E. coli

• Cells are first lysed
• Protein extract is subjected to affinity
  chromatography on GSH-sepharose GST
  (Glu-Cys-Gly) binds GSH and is retained on the
  column
• 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.

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 organismsSource Genetic
Engineering News. 2004, 24(18) 22-28.

• Bacteria
• Advantages
• 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.

• Yeast
• Advantages
• Eukaryote allows for post-translational
  modifications
• Rapid growth
• High yield
• Inexpensive to scale up
• Secretes products into the medium easily
• Splices pre-mRNA
• 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
• 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
• Zeocin
• an antibiotic
• allows for selection of yeast containing the
  vector
• C-myc epitope
• Amino acid sequence from c-myc protein
• Used for easy detection by Western blot (reviewd
  laterpatience!)
• a-factor
• Mating factor secreted by yeast cells
• Allows for secretino of the foreign protein into
  the culture media
• 6 x HIS
• 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 Ni2 is bound
• The His residues of the tag will bind the Ni2,
  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
• 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

• Mammalian cells
• Advantages
• 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
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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
• 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

• Mammalian cells
• Advantages
• 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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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
Thr(103)?Asn Asn (117)?Gln Lys-His-Arg-Arg (296-299)? Ala-Ala-Ala-Ala 8.3 0.87 0.85
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Mammalian expression vector
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Mammalian Promoters
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Useful Mammalian Promoters

• Viral promoters
• Constitutive
• CMV
• SV40
• Inducible
• MMTV (glucocorticoids)

• Cellular promoters
• Constitutive
• Ubiquitin (UbC)
• Thymidine kinase
• Human eIF1a
• 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 transfectionStable vs transient
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Transfection procedures
Calcium Phosphate precipitation
Electroporation
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 transductionRetroviruses
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Cell transductionRetroviral 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)
• Integration is random and can lead to insertional
  mutagenesis insertion in the genome may lead to
  cancer.

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Cell transductionRetroviruses
http//www.ncbi.nlm.nih.gov/books/bv.fcgi?ridrv.s
ection.4357
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Cell transductionRetrovirus production
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Cell transductionAdenoviruses

• Adenovirus
• 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)

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Cell transductionAdenoviruses
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Cell transductionAdenoviruses
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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 proteinGreen 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 proteinGreen fluorescent Protein (GFP)
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Fusion proteinGreen fluorescent Protein (GFP)
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Fusion proteinGreen fluorescent Protein (GFP)
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Fusion proteinGreen fluorescent Protein (GFP)
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Fusion proteinGreen fluorescent Protein (GFP))
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Fusion proteinGreen fluorescent Protein (GFP)