Transcript 8 Expression and Modification of Recombinant Proteins

Chapter 8: Expression and
Modification of Recombinant
Proteins
Prokaryotic expression
system
Eukaryotic expression system
I.
II.
1.
2.
3.
Yeast expression
Insect expression system
Mammalian expression system
References
Chapter 11 and 13 (Essentials
of Molecular Biology)
2. Chapter 10 (An Introduction to
Genetic Engineering)
1.
I. Prokaryotic
Expression System
How do you express this gene in
bacteria?
Insert only the ORF:
The red boxes
represent exons, the
blue boxes represent
the introns, and the
grey boxes represent
the 5’ and 3’ UTRs
into an expression vector
that contains prokaryotic transcriptional and
translational regulatory sequences
Six Step Process
①
②
③
④
⑤
⑥
Isolation of gene of interest
Introduction of gene to expression
vector
Transformation into host cells
Growth of cells through fermentation
Isolation & purification of protein
Formulation of protein product
Cloning Process
Gene of interest is cut
out with restriction
enzymes (RE)
* Host plasmid (circular
chromosome) is cut
with same REs
* Gene is inserted into
plasmid and ligated
with ligase
* New (engineered)
plasmid inserted into
bacterium (transform)
*
Fundamentals of Gene
Expression
①
Prokaryotic and eukaryotic
promoters and translation signals
are different...they are not
exchangeable
You therefore can’t simply put a
eukaryotic promoter into bacteria and
expect it to function
②
Processing also presents a problem for
bacterial expression of human mRNAs.
The eukaryotic ORF must be cloned into the
expression vector, not the genomic copy
②
The genetic code is identical for most
bacterial and eukaryotic mRNAs.
Although the code is the same, expression levels
can be affected by codon frequency, which varies
between organisms and transcripts
④
Post-translational modifications can be
important for protein function
Those modifications might not occur in bacteria.
The solution…try expressing in a eukaryotic
expression system (viral, baculovirus, yeast)
Prokaryotic Expression
Pros
Convenient/Easy
Produce and purify protein using the least
expensive and easiest reagents and equipment
* Best for large scale production of protein
*
*
Cons
*
*
*
lack many of the immunogenic properties
3D “Native” conformation
Lack PTMs (Post translational modifications)
needed for specific activity
Which Vector?
*
Promoters
arabinose systems (pBAD), phage T7
(pET), Trc/Tac promoters, λ PL or PR
Tags标签
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*
*
*
*
*
*
His6 for metal affinity chromatography (Ni)
FLAG epitope tage DYKDDDDK
CBP-calmodulin binding peptide (26 residues)
E-coil/K-coil tags (poly E35 or poly K35)
c-myc epitope tag EQKLISEEDL
Glutathione-S-transferase (GST) tags
Celluluose binding domain (CBD) tags
Fusion Protein
Gene
T7 Promoter
RBS
lac Operator
( with / without stop codon )
ATG
TAG
peptide tag: V5 / poly-(his)
mutiple cloning site (mcs)
peptide tag: V5 / poly-(his)
A Generic Vector
II. Eukaryotic Expression
System
1.
Yeast expression
2.
Insect expression system
3.
Mammalian expression system
1. Yeast Expression
Pros
*
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*
*
*
*
Easier and less expensive than higher eukaryotic cells
Rapid growth on inexpensive media
Ideal for large-scale production of heterologous
proteins
Often comfortable with genetic manipulation
Exhibit near-native conformation, PTMs, processing
Well-defined secretory pathways for extracellular
export of the recombinant gene product
Usually safe to use
Cons
*
*
*
Lack some PTMs required for specific activities
A little more expensive than prokaryote systems
Often lack mechanisms for proper folding for some
eukaryote proteins
Yeast Expression Vector
Comments for pPIC9K: 9276 nucleotides
5´ AOX1 promoter fragment: bases 1-948
5´ AOX1 primer site: bases 855-875
a-Factor secretion signal(s): bases 949-1218
a-Factor primer site: bases 1152-1172
Multiple Cloning Site: bases 1216-1241
3´ AOX1 primer site: bases 1327-1347
3´ AOX1 transcription termination (TT):
bases 1253-1586
HIS4 ORF: bases 4514-1980
Kanamycin resistance gene: bases 5743-4928
3´ AOX1 fragment: bases 6122-6879
pBR322 origin: bases 7961-7288
Ampicillin resistance gene: bases 8966-8106
Pichia pastoris
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*
Yeast are single celled eukaryotes
Behave like bacteria, but have key
advantages of eukaryotes
P. pastoris is a methylotrophic甲醇为营养的
yeast that can use methanol甲醇as its
sole carbon source (using alcohol
oxidase)
Has a very strong promoter for the
alcohol oxidase (AOX) gene (~30% of
protein produced when induced)
Cloning in Yeast Cells
①
②
③
④
Uses a special plasmid that works both in
E.coli and Yeast
Once gene of interest is inserted into this
plasmid, it must be linearized (cut open so it
isn’t circular)
Double cross-over recombination event
occurs to cause the gene of interest to
insert directly into P. pastoris chromosome
where the old AOX gene used to be
Now gene of interest is under control of the
powerful AOX promoter
Cloning
1
2
3
4
2. Insect Expression
Pros
*
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Produce proteins that has PTMs similar to
mammalian systems
Often properly folded and functional
Ideal for producing moderate to high levels
of eukaryotic proteins for structure-function
assays
Cons
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Expensive
Sometimes proteins are not correctly folded
Often not stable
Autographica californica multiple
nuclear polyhedrosis virus
(Baculoviurs)
* Virus commonly infects insects cells
of the alfalfa looper (small beetle) or
armyworms (and their larvae)
* Uses super-strong promoter from the
polyhedron coat protein to enhance
expression of proteins while virus
resides inside the insect cell
*
Baculovirus (AcMNPV)
Cloning Process
Transfer vector
Cloned gene
5’
3’
x
x
Cloned gene
5’
3’
Polyhedrin gene
AcMNPV DNA
Recombinant
AcMNPV DNA
Baculovirus
BacVirus Expression System
*
*
pIEx™Insect Cell
Expression
Plasmids (Novagen)
Bac-NBlue™Baculovirus
Expression System
(Invitrogen)
3. Mammalian Expression
Pros
*
*
Produce protein in the most native and active form
Have required PTM machinery to produce active
and useable protein used in mammals
Cons
Expensive
Unstable
Low yield and difficulties in purifying recombinant
proteins
* Limitations on the mechanisms of protein
expression induction
* Almost always have over expression
*
*
*
HeLa
cells in
culture
HeLa cells from
the Nikon
microscope web
site
HeLa cells have been cultured
continuously for scientific use
since they were first taken
from the tumor of a woman
suffering from cervical cancer in
the 1950s. They have
been utilized for many purposes,
including the development of a
polio vaccine, the pursuit of a
cure for diseases such as
leukemia and cancer, and the
study of the
cellular effects of drugs and
radiation.
HeLa Human cells
JONATHON PINES
REGULATION OF MITOSIS IN
MAMMALIAN CELLS
Mitotic HeLa cell stained with
anti-Cks1 (red), anti-tubulin
(yellow) and DAPI (blue)
HeLa cells as you will see them
Expression of (A) b-galactosidase and B green fluorescent protein
in HeLa cells. Cells were transfected in 6-well plates. Expression
was visualized by X-gal staining or fluorescence microscopy 2
days post-transfection.
Expression System
Selection
Choice depends on size and character of
protein
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Large proteins (>100 kD)? Choose eukaryote
Small proteins (<30 kD)? Choose prokaryote
Glycosylation essential? Choose baculovirusor
杆病毒 mammalian cell culture
High yields, low cost? Choose E. coli
Post-translational modifications essential?
Choose yeast, baculovirus or other eukaryote
Which Vector
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Must be compatible with host cell system
(prokaryotic vectors for prokaryotic cells,
eukaryotic vectors for eukaryotic cells)
Needs a good combination of
*
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strong promoters
ribosome binding sites
termination sequences
affinity tag or solubilization sequences
multi-enzyme restriction site
Key Parts to a Vector
Origin of replication (ORI) – DNA sequence for
DNA polymerase to replicate the plasmid
* Selectable marker (Amp or Tet) – a gene, when
expressed on plasmid will allow host cells to
survive
* Inducible promoter – Short DNA sequence
which enhances expression of adjacent gene
* Multi-cloning site (MCS) – Short DNA sequence
that contains many restriction enzyme sites
*
Mammalian Expression
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*
Gene initially cloned and plasmid propagated
in bacterial cells
Mammalian cells transformed by
electroporation (with linear plasmid) and gene
integrates (1 or more times) into random
locations within different CHO chromosomes
Multiple rounds of growth and selection using
methotrexate to select for those cells with
highest expression & integration of DHFR and
the gene of interest
Methotrexate (MTX)
Selection
Gene of interest
DHFR
Transfect
dfhr- cells
Grow in
Nucleoside
Free medium
Culture a
Colony of
cells
Grow in
0.05 uM Mtx
Culture a
Colony of
cells
Methotrexate (MTX)
Selection
Grow in
0.25 uM Mtx
Culture a
Colony of
cells
Grow in
5.0 uM Mtx
Culture a
Colony of
cells
Foreign gene
expressed in
high level in
CHO cells
Mammalian Expression
Systems
Comments for pcDNA™4/TO : 5078 nucleotides
CMV promoter: bases 232-958
TATA box: bases 804-810
Tetracycline operator (2X TetO2) sequences: bases
820-859
CMV forward priming site: bases 769-789
Multiple cloning site: bases 967-1077
BGH reverse priming site: bases 1089-1106
BGH polyadenylation sequence: bases 1095-1319
f1 origin: bases 1365-1793
SV40 promoter and origin: bases 1803-2143
EM-7 promoter: bases 2183-2249
Zeocin™ resistance gene: bases 2250-2624
SV40 early polyadenylation sequence: bases 27542884
pUC origin: bases 3267-3937
bla promoter: bases 4937-5041 (complementary
strand)
Ampicillin (bla) resistance gene: bases 4082-4942
(complementary strand)
Mammalian Cell-line
Expression
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*
*
Sometimes required for difficult-to-express
proteins or for “complete authenticity”
(matching glycosylation and sequence)
Cells are typically derived from the Chinese
Hamster Ovary (CHO) cell line
Vectors usually use SV-40 virus, CMV or
vaccinia virus promoters and DHFR
(dihydrofolate reductase) as the selectable
marker gene
Summary
1.
2.
3.
4.
The mechanism of regulation of gene
expression in prokaryotes is greatly different
from eukaryotes
Regulation of gene expression in prokaryotes
is mainly for environmental adaptation
Regulation of gene expression in eukaryotes
is for cell growth, differentiation and
development
There are four recombinant protein
expression systems: bacteria (E.coli), yeast,
insect and mammalian cells. Choose by
purpose