LECTURE 15
EUKARYOTIC EXPRESSION VECTOR
LY N N C A L L I S O N
CHEMICAL BIOLOGY
DR. KUANG-YU CHEN
RUTGERS UNIVERSITY
I.
OVERVIEW
II. RECOMBINANT GENE EXPRESSION
III. OPERON CONTROLS GENE EXPRESSION
IV. EUKARYOTIC EXPRESSION SHUTTLE
VECTOR
V. TAGGING SEQUENCE OVERVIEW
A. POLYHISTIDINE AND EPITOPE
TAGS
B. GREEN FLUORESCENT PROTEIN
TAG
VI. TRANSFECTION TECHNOLOGY
OVERVIEW
A. CHEMICAL TRANSFECTION
B. PHYSICAL AND BIOLOGICAL
TRANSFECTON
VII. CURRENT NEWS: GENE THERAPY
VIII.
WORKS CITED
1
OVERVIEW
An expression vector is used to express a
cloned gene in a host cell. The vector contains
regulatory sequences that direct the host cell to
transcribe the foreign gene into messenger
RNA (mRNA). The resulting RNA is then
translated into protein (fig1). Expression vectors
are prepared as plasmids, phages, or
phagemids depending on the desired efficiency.
For expression of human proteins, the vector
requires regulatory sequences such as
promoters that recruit the RNA polymerase in
both prokaryotes and eukaryotes.
Human
expression vectors begin in bacteria host cells
for ease of use and end in yeast host cells for
eukaryotic post translational modifications. The
cloned gene is often engineered to contain an
additional sequence that allows it to become
purified and quantified in the laboratory. Figure2
depicts a protein that was cloned with an
attached green fluorescent protein (GFP)
sequence. Expression vectors are useful for the
development of medical treatments. These
vectors have been used to prepare in vitro
human insulin for the treatment of diabetes.
Expression vectors are designed to produce Figure 2:Image of structural proteins bound to
large amounts of one desired segment of GFP
mRNA and resulting protein on command. (4, 12, 13)
Figure 1: Overview of gene expression vector. Numerous processes are required for preparation of
both the target DNA and target protein
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RECOMBINANT GENE EXPRESSION
Expression vectors are prepared by standard cloning techniques. The expression vector target
DNA is prepared from target mRNA (fig3). The cell transcribes DNA to RNA and then translates
RNA to protein. If the amino acid sequence of the desired target protein is known, the genetic
code can be used to translate the amino acid sequence to a nucleotide sequence. From this
nucleotide sequence, a complementary radioactively labeled probe can be prepared to identify
the complete mRNA in the midst of a cell’s contents. Once the complete protein encoding
mRNA is obtained, Reverse transcriptase uses the mRNA sequence to generate a
complementary DNA (cDNA) sequence. The RNAse enzyme is used to digest the
phosphodiester back bone of the mRNA. Once the mRNA is removed from the single stranded
cDNA, reverse transcriptase is used to prepare a double stranded cDNA segment. For
introduction into the vector, ligase enzyme is used to attach synthetic ‘linker’ sequences to the
ends of the double stranded cDNA. These ‘linkers’ supply ‘sticky-end’ sequences for annealing
with a restriction enzyme splice site. Ligase is used again to attach the cDNA to the ‘cloning site’
of the vector. The ‘cloning site’ consists of an assortment of endonuclease splice site. It supplies
a source of potential ‘sticky ends’ for the attachment of a target gene. The ‘Splice site’ is
strategically located downstream of regulatory sequences. The promoter regulatory sequence
recruits RNA polymerase to the site. RNA polymerase binding causes all of the downstream
DNA sequences to be transcribed into RNA. The adjacent ribosome binding regulatory
sequence causes the transcribed RNA to contain a nucleotide sequence for ribosome
attachment and enables initiation of translation of RNA into protein. For the purpose of efficient
protein expression in a host cell, each vector must contain a replication origin (ori) for
recruitment of DNA polymerase when the DNA is replicated during cell division. Additionally,
each vector has an antibiotic resistance gene (Ampi) to enable scientists to colonize host cells
on antibiotic rich medium and identify the cells that have successfully incorporated the vector
and have high probability of producing gene product target protein. (9, 13)
Figure 3: (left) Procedure for preparation of target DNA in cloning vector (right) regulatory sequences
required to enable the vector to form target protein
3
OPERON CONTROLS GENE EXPRESSION
An operon is a genetic regulatory element that regulates gene expression. The operon
sequence controls whether the target gene is constantly translated into protein (constitutive) or if
it is triggered into making protein in command by the presence of a stimulating molecule
(induced). For the purpose of laboratory efficiency, expression vectors are inducible, also
known as regulated. The basic expression vector contains a replication origin (Ori), a selectable
antibiotic-resistance gene, and a strong-regulated promoter. This section uses the lac operon to
discuss the means by which the operon regulates and induces the promoter. (2)
The lac operon consists of a repressor structural gene (lacI), promoter (P), operator (O),
structural genes (Z, Y, and A), and transcription/translation termination sequences (fig4). The
promoter is a DNA sequence in the operon that RNA polymerase binds to transcribe the DNA
sequence into RNA sequence. The operon is repressed by binding of the lac repressor enzyme
(encoded at I) to the operon (O). Constituently transcribed and translated, the lac repressor
enzyme prevents transcription by binding at the operon and preventing the forward movement
of RNA polymerase. In the presence of lactose, the lac repressor enzyme disassociates from
the operator. The RNA polymerase then proceeds to transcribe the Z, Y, and A structural genes.
The Z, Y, and A structural genes are separated by ribosome start and stop codons. This
enables the ribosome to translate three separate enzymes that promote lactose metabolism.
LacZ, encodes the enzyme, β-galactosidase for hydrolysis of lactose to glucose and galactose.
LacY, produces permease, for the transport of lactose into the cell. lacA, codes for
transacetylase, whose role in lactose metabolism is unknown. Overall, the expression of lactose
metabolism enzymes is “switched on” (regulated) by the presence of lactose. (1, 2)
The structure of the lac operon is frequently used to regulate the expression vector promoter.
Inducible expression such as that observed in the presence of lactose in the lac operon is
beneficial for two reasons. Firstly, high levels of a foreign protein can be toxic to the host cell.
Secondly, expression of a foreign protein at a constant level can sequester valuable host cell
energy and prevent the population of host cell from growing to enough quantities to enable a
sufficiently large harvest of protein product. In the laboratory, scientists use the lac operon
concept by constructing a vector that carries the lacI repressor gene upstream of a promoter,
operator and target gene for desired protein. In this way, scientists can supply or deprive the
host cell of lactose (or synthetic analog, IPTG (Isopropyl β-D-1-thiogalactopyranoside)) to turn
“on” and “off” transcription of the gene for the target protein. (4)
Figure 4:Diagram of the Lac Operon as it is transcribed into mRNA and translated into protein
4
EUKARYOTIC EXPRESSION SHUTTLE VECTOR
Expression vectors produce the target protein of a cDNA gene. They
consist of an origin of replication, promoter, postranslaitonal
modification signals, antibiotic resistance genes, and the cDNA target
gene. They utilize, strong inducible promoter to produce the largest
possible amount of target protein on an application of a chemical
stimulus (e.g. IPTG). Furthermore, eukaryotic expression vectors are
also known as shuttle vectors. Shuttle vectors enable efficient cloning
and optimum amount of active target protein/enzyme production. (1,
2)
Protein expression vectors are also known as shuttle vectors. Shuttle
vectors can replicate in both prokaryotes and eukaryotes and must
possess an origin of replication for each. Although prokaryotic cells
are more convenient for use in initial cloning and transformation steps,
eukaryotic host cells apply post translational modifications that
produce cloned human proteins of high enzymatic activity. When
cloning eukaryotic proteins, eukaryotic host cells cause the
posttranslational modifications of phosphorylation and glycosylation
that are specific to eukaryotes. Furthermore, eukaryotic host cells
possess an intercellular environment that optimizes protein folding
and minimizes aggregation. Overall, shuttle vector cloning sequences
are optimized in prokaryotes and are then transferred to eukaryotic
cells to harvest a eukaryotic protein of optimal activity. A eukaryotic
protein expression vector (fig5) must contain: (1, 2, 7)
Figure 5: Schematic of Eukaryotic
Expression Vector
1. Prokaryotic and Eukaryotic Origin of Replication
 f1 origin (ori) Allows rescue of single-stranded DNA S. cerevisiae (eukaryote)
 SV40 origin (ori) Allows efficient, high-level expression of the neomycin
 resistance gene and episomal replication in cells expressing SV40 large T antigen
 Puc origin (ori) High-copy number replication and growth in E. coli (prokaryote)
2. Strong inducible promoter up stream of a multiple cloning site (MCS)
 SV40 early promoter (PSV40) Allows efficient, high-level expression of the neomycin
 resistance gene and episomal replication in cells expressing SV40 large T antigen
 Human cytomegalovirus (PCMV) immediate-early promoter/ enhancer–Permits
efficient, high-level expression of your recombinant protein
 T7 promoter/priming site ---Allows for in vitro transcription in the sense orientation and
sequencing through the insert
 Multiple cloning site in forward or reverse orientation---Allows insertion of your gene
and facilitates cloning
3. Posttranslational modification signals
 Bovine growth hormone (BGH PA) polyadenylation signal (PA) Efficient transcription
termination and polyadenylation of Mrna
 SV40 early polyadenylation signal (SV40 PA) Efficient transcription termination and
polyadenylation of Mrna
4. Antibiotic resistance gene
 Neomycin resistance gene Selection of stable transfectants in eukaryotic cells
 Ampicillin resistance gene(β-lactamase)–Selection of vector in prokaryotic cells
5. Target gene Cdna to encode complete eukaryotic target protein
5
TAGGING SEQUENCE OVERVIEW
Once the vector is cloned and successfully transcribes mRNA, the
resulting target protein is purified through tagging techniques. The
tag consists of an additional amino acid sequence that improves
purification through intercellular signaling, green fluorescent protein
(GFP) fluorescence, metal ion coordination, or antibody recognition.
In the laboratory a tag is added during vector preparation by
combining the gene sequence for target protein with that of the
tagging protein without an intervening ribosome stop command
(fig6). The result is one long peptide that has target protein and
tagging protein features. (4, 13)
Naturally fused amino acid targeting sequences are present in cells
to divert proteins to the necessary organelles. They can send
proteins to the endoplasmic reticulum, mitochondria, chloroplast,
peroxisome, or nucleus. These amino acid sequences are listed in
figure 7. Also known as a topogenic tag, this sequence of residues
ensures that the peptide is in the correct orientation when
incorporated into the organelle plasma membrane. In the laboratory,
scientists utilize the endoplasmic reticulum or golgi apparatus
targeting sequences to cause the localization of target proteins to
secretory vesicles. By initiating the secretion of the cloned target
protein with a targeting tag, scientists can easily harvest and
separate the protein from other cellular biomolecules. (4, 9)
Figure 6:Transcription and translation of
fusion protein
Figure 7: Targeting sequences that direct proteins to organelles
Target
Location in
Organelle
Protein
Nature of Signal
“Core” of 6–12 mostly hydrophobic amino acids, often preceded by one or more basic amino
Endoplasmic
N-terminal
reticulum
acids
Mitochondrion
N-terminal
Chloroplast
N-terminal
3 – 5 nonconsecutive Arg or Lys residues, often with Ser and Thr; no Glu or Asp residues
No common sequence motifs; generally rich in Ser, Thr, and small hydrophobic amino acid
residues and poor in Glu and Asp residues
Peroxisome
C-terminal
Nucleus
Internal
Usually Ser-Lys-Leu at extreme C-terminus
One cluster of 5 basic amino acids, or two smaller clusters of basic residues separated by
≈10 amino acids
6
POLYHISTIDINE AND EPITOPE TAGS
A polyhistidine tag is useful for purification of
the cloned protein. The tag is a sequence of
approximately five histidine residues translated
at the N-terminal end of the target protein. It is
prepared in the vector by including the
polyhistidine sequence upstream of the cloned
target protein without an intervening ribosome
stop sequence. The polyhistidine tag enables
purification by affinity chromatography with
nickel ion (Ni2+) bound sepharose gel (fig8).
After the host cells are collected and lysed, the
target protein is released for purification. The
nickel ion of the sepharose coordinates with the
histidine residues and causes the target protein
to stay on the column. It is released when the
column is rinsed with a high concentration of
histidine residues that act as competitive ligand
for the nickel ion. The tagged target protein is
then separated from the polyhistidine via
cleavage at a enterokinase sequence. A final
affinity column purifies the mixture by removing
the polyhistidine tag. (13)
Antibody recognition of epitope tagging is
used for recombinant protein purification. An
epitope is the portion of an antigen that is
recognized
by
antibodies.
Eukaryotic
expression vector DNA is prepared to include
coding for a common epitope. Common
epitopes are recognized by antibodies that can
be obtained commercially. Common epitopes
are short protein sequences derived from full
proteins (fig9). This system allows light or
electron microscope analysis via use of an
epitope-atibody in conjunction with a labeled
secondary antibody (fig10). Furthermore, the
epitope tagged protein can be purified by
immunoprecipitation,
immunoaffinity
chromatography, or isolated in a colony. (13)
Figure 10: Colony isolation of peptide bearing an epitope
tag
Figure 9:Common epitope tag sequences
Tag
HIS
c-MYC
Figure 8: Affinity column purification of peptide with
polyhistidine tag
HA
VSV-G
HSV
V5
Sequence HHHHHH EQKLISEEDL YPYDVPDYA YTDIEMNRLGK QPELAPEDPED GKPIPNPLLGLD
7
GREEN FLUORESCENT PROTEIN TAG
Green fluorescent protein (GFP) is used as a fluorescent tag fused to the target protein. The
location of the GFP fusion protein can be tracked via fluorescence microscopy the in a in a living
or dead cell. Localization of the target protein provides information about its function. The fusion
protein is prepared in the expression vector by placing the GFP upstream of the target protein
without an intervening ribosome stop sequence (fig11A). Originally found in jelly fish, GFP
consists of 238 residues which make up a β-barrel structure that surrounds the chromophore
residues: Tyr66 and Ser65 (fig11B). GPF is photoactivated by an exposure to 405nm light that
causes decarboxylation of Glu222 and excitation of the pi-electron clouds of Tyr66 and Ser65
(fig11C). This light emission is observed by fluorescence microscopy and allows scientists to
observe the location of the target protein in the cell. (7, 11, 13)
(A)
(B)
(C)
Figure 11:(A)Vector diagram for a target protein that contains a GFP tag , myc epitope tag, and three
repeated sequence of nuclear targeting sequence (B) Rendered image of GFP (C) Photoactivation
reaction of essential residues in GFP
8
TRANSFECTION TECHNOLOGY OVERVIEW
Introduction of foreign DNA into eukaryotic cells is called transfection. Cells exposed to DNA are
coerced into taking up the DNA. Normally, the foreign DNA vector is a transient transfection and
is only temporarily present in the host cell population. Transient transfection is useful if the
protein gene produce can be harvested quickly. It supplies a temporary, high level of gene
expression for approximately 1 to4 days following transfection. However, if laboratory
procedures require a cell host cell that can dispense cloned gene product for a long period,
stable transfection is necessary. Stable transfection occurs when the cloned gene is
incorporated and expressed in the host genome and retained with each cell division cycle. This
occurs at a <0.1% frequency. The stably transformed cells are identified through use of a
selectable marker. A selectable marker is a genetic sequence that is included in the vector that
gives the host cell an identifiable trait such as drug resistance or metabolomic capabilities.
Several common selectable markers are listed in figure 12. The pathway of stable transfection
in figure13, shows that the circular vector DNA contains the target gene in red and the
selectable marker in blue is integrated into the linear host genome during the last step. Note that
calcium phosphate is used to coprecipate the DNA during the initial transfection phase. Calcium
phosphate is one type of several chemical, physical or biological methods of transfection. (4)
Figure 12: Selectable Markers for Stable Transfection





Aminoglycoside phosphotransferase (APH; neoR gene) – Neomycin resistance
Adenosine dreaminase (ADA) – enzyme involved in purine metabolism. It is needed for the
breakdown of adenosine from food and for the turnover of nucleic acids in tissues.
Dihydrofolate reductase (DHFR) – enzyme that reduces dihydrofolic acid to tetrahydrofolic
acid, using NADPH as electron donor,
Thymidine kinase (TK) – enzyme with a key function in the synthesis of DNA and cell
division, as they assist the introduction of deoxythymidine into the DNA.
Xanthine-guanine phosphoribosyl transfersase (XGPRT; gpt gene)- a purine salvage
enzyme
Figure 13:The process of stable transfection; beginning at the vector and ending at the cell
9
CHEMICAL TRANSFECTION
Chemical transfection of expression vectors into
eukaryotic cells is mediated by calcium phosphate,
liposome,
DEAE-dextran,
or
dendrimers,
Coprecipitation of the DNA with calcium phosphate
is an inexpensive method of chemical transfection.
Calcium phosphate is shown in step one of
figure13, and as a structure in figure14A. The
mechanism of calcium phosphate transfection is
not completely understood. However it is believed
that a precipitate of calcium phosphate salt Ca3
(PO4)2 promotes adherence DNA to the surface of
the crystal. This DNA coated salt is then applied to
the host cell, causing it to take up a portion of the
foreign DNA. Lipofection, or liposome mediated
transfection, is another common method of
chemical transfection. The molecules of lipofection
consist of a cationic head and a hydrocarbon tail
(fig14B). They are similar in structure to the
phospholipids of the cell membrane. They function
by surrounding the foreign DNA and facilitating its
fusion into the cell membrane. Once the complex is
associated with the host cell membrane, the DNA is
released into the host cell. Diethylaminoethyldextran (DEAE-dextran) functions similarly to
lipofection. As shown in figure14C, DEAE-dextran
possesses a sugar chain linked by an O-glycosidic
bond to a cationic tail. The negatively charged DNA
binds to the cationic tail. The DNA bound molecule
possesses excess cationic activity and uses this
polarity to become enmeshed in the phospholipids
bilayer. Evidence suggests that the dextran-DEAEDNA complex enters the cell via endocytosis.
Dendrimer technology further elaborates on the
chemical aggregation method of DNA transfection.
As shown in figure14D, dendrimers are branched
organic molecules with polar functional groups
bound to the end of each branch. The structure is
synthesized via a series of substitution reactions. It
is engineered with the goal of encapsulating DNA
molecules. The positively charged amino group at
each branch of the dendrimer is designed to
interact with the negatively charged phosphate
group of DNA. Overall, the chemical method of
transfection exploits the polar nature of DNA to
form a chemical aggregate that is compatible with
the hydrophobic and hydrophilic interactions of the
phospholipids bilayer. (4, 14)
A)
B)
C)
Dextran
D)
Figure 14: Chemical Methods of Transfection (A)
Calcium phosphate (B) Liposomes (C) DEAEdextran (D) Dendrimer
10
PHYSICAL AND BIOLOGICAL TRANSFECTION
Physical transfection methods employ
scientific instruments to introduce
foreign DNA into a eukaryotic cell.
Physical
methods
include
electroporation,
ballistics,
and
microinjection. Electroporation is the
application of an electric field to a
mixture of host cells and foreign DNA.
The electrical field is believed to
disrupt the phospholipids bilayer of the
host cell and permit access to the
foreign DNA. The left side of figure15A
depicts the electroporation apparatus.
The right side of the figure shows an
idealized membrane pore formation.
However, the exact structure of the
membrane pores resulting from
electroporation cannot be confirmed.
A ballistic particle delivery system, or
gene gun, is able to transform
eukaryotic cells as well as prokaryotic
and plant cells (fig15B). It “injects”
DNA by propelling a DNA-coatedmetal-particle into the target cell. Gene
guns can introduce DNA to any part of
the host cell’s nucleus or organelles.
In addition to cloning of eukaryotic
expression vectors, microinjection is
used for cloning of an entire organism.
It uses a glass micropipette to insert
DNA into the nucleus of a single living
cell. This procedure requires a
“specialized
optical
microscope”,
holding-pipette, and micropipette of
0.5-5.0
Μm
diameter
(fig15C).
Physical transfection methods require
expensive equipment and are typically
used after chemical transfection has
proved ineffective. (14)
A)
B)
C)
Figure 15: Physical Transfection
(A) Electroporation (B) Gene gun
(C) Microinjection
Figure 16:
Biological Transfection:
lentiviral vector
Biological transfection, also known as infection uses lentiviral vectors to introduce cloned DNA
into eukaryotes. A lentiviral vector is developed by removing the pathogenesis genes from a
retrovirus. They are very efficient for biological transfection in eukaryotic cells. Lentiviral vectors
are useful for stable transfection because of the enzyme, integrase. After infecting the host cell,
integrase functions to incorporate the foreign DNA into the DNA of the host’s genome. Figure 16
shows that retroviral vectors are cloned in host cells and can then be isolated as virus particles
for an infection that results in a stable transfectant Because of its efficiency, retroviral vectors
have use in gene therapy. Gene therapy is the introduction of foreign genes to correct
malfunctioning disease causing genes. (4, 13)
11
CURRENT NEWS: GENE THERAPY
LENTIVIRAL EXPRESSION VECTOR FOR PARKINSON’S DISEASE
Conclusive evidence indicates that lentiviral vectors can be used to treat the symptoms of
Parkinson's disease. These symptoms are caused by degeneration of the dopaminergic
neurons of the substantia nigra (SN) section of the basal ganglia in the brain. Glial cell linederived neurotrophic factor (GDNF), acts as a neuroprotective agent that can treat the diseased
neuron cells of Parkinson's disease. Scientists prepared a lentiviral expression vector that is
designed to produce GDNF (lenti-GDNF)in the SN.For experimental purposes, the disease
model is prepared by injecting adult monkeys with the compound,1-methyl-4-phenyl-1,2,3,6tetrahydropyridine (MPTP), to cause motor defects. The experiment proceeded by injecting the
monkeys with lentiviral vectors for GDNF or β-galactosidase as a control. The monkeys were
then subjected to a hand-reach task (fig17) before biopsy (fig18). During the hand-reach task,
lenti-GDNF treatment was shown to improve the reactivity of MPTP monkeys. Furthermore,
biopsy indicates that lenti-GDNF prevented SN degradation. (8)
Figure 17: Results of hand-reach task shows lenti-GDMF treated monkies perform better up
to 3 months
Figure 18:SN neuron image of (C) more neurons in lenti-GDNF treated subject and (D) less
neurons in untreated subject.
12
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Berg, J. et. al. Biochemistry, 5th ed. W H Freeman and Company, New York, (2002) 27.2.
(2)
Campbell, et. al. Biology, 5th ed. Addison Wesley Longman, Inc., New York, (1999) 284293.
(4)
Cooper, G. M. The Cell - A Molecular Approach, 4th ed. Sinauer Associates, Inc,
Sunderland MA, (2007) 201-226.
(5)
Garett and Grisham. Biochemistry 2nd ed. Thomson: Brooks/Cole: United States (1999)
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(7)
Invirogen: life technologies. “pcDNA” 2010, Mon 3 May < http://www.invirogen.com>
(8)
Kordower, J H. et al. "Neurodegeneration Prevented by Lentiviral Vector Delivery of GDNF
in Primate Models of Parkinson's Disease." Science 290, 767-773 (2000).
(9)
Lodish et. al. Molecular Cell Biology. W H Freeman and Company, New York, (1999) 11.6.
(10) Papale A. “Viral vector approaches to modify gene expression in the brain” Journal of
Neuroscience Methods 185, 1-14 (2009) 1–14.
(11) Runions, John, et. al. "Photoactivation of GFP reveals protein dynamics within the
endoplasmic reticulum membrane" Journal of Experimental Botany 2006 57(1):43-50
(12) Strachan, T. et. al. Human Molecular Genetics, 2nd ed. Garland Science, New York,
(1999) 22.3.2.
(13) Weaver, Robert F. Molecular Biology, 4th ed. McGraw Hill Higher Education: New York,
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(14) Wikipedia 2010, Mon 3 May “Transfection” <http://en.wikipedia.org/wiki/Transfection >
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