Title: Recombinant DNA Technology CHMI 4226 E
1Recombinant DNA TechnologyCHMI 4226 E
- Week of March 14, 2005
- Expression of proteins into living organisms
2Expression of cDNAs
3Why express recombinant proteins?
4Expression 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
5Expression 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
6Expression 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.
7Expression 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.
8pGEX vector
9Expression in E. coli Cloning in pGEX
Added restriction sites
Eliminate STOP codon
ORF of YFcDNA in frame with GST
OR
10Expression 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
11Expression in E. coli
12Expression 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
13Expression 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
14Expression 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!).
15Expression 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.
16Protein 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).
17Expression 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
18Using insect cells for protein expression the
baculovirus system
19Using insect cells for protein expression the
baculovirus system
20Using insect cells for protein expression the
baculovirus system
21Using 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.).
22Using insect cells for protein expression the
baculovirus system
23Using 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
24Using insect cells for protein expression
25Recombinant 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
26Expression 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
27Protein 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
28Protein 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
29Mammalian expression vector
30Mammalian Promoters
31Useful 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
32Antibiotic resistance genes
33Optimization of translation in mammalian cells
- Kozak consensus sequence
- (GCC)GCCA/GCCATGG
- Ensures optimal translation initiation
- Can easily be added by PCR-mediated mutagenesis.
34Epitope 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
35Mammalian cell transfectionStable vs transient
36Transfection procedures
Calcium Phosphate precipitation
Electroporation
Lipofection
37Cell transduction
- Viruses are widely used to introduce DNA
molecules into mammalian cells - Form the basic vehicle for gene therapy
38Cell transductionRetroviruses
39Cell 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.
40Cell transductionRetroviruses
http//www.ncbi.nlm.nih.gov/books/bv.fcgi?ridrv.s
ection.4357
41Cell transductionRetrovirus production
42Cell 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)
43Cell transductionAdenoviruses
44Cell transductionAdenoviruses
45Transfection 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).
46Fusion 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!
47Fusion proteinGreen fluorescent Protein (GFP)
48Fusion proteinGreen fluorescent Protein (GFP)
49Fusion proteinGreen fluorescent Protein (GFP)
50Fusion proteinGreen fluorescent Protein (GFP)
51Fusion proteinGreen fluorescent Protein (GFP))
52Fusion proteinGreen fluorescent Protein (GFP)