Exprssion vector

A Presentation on
Expression
Vector
Presented By –
Sushant Balasaheb Jadhav
Roll no. – 18PBT206
M. Tech Pharmaceutical Biotechnolgy
Institute of Chemical Technology,
Mumbai-400019

A vector used for expression of a cloned DNA fragment in a host
cell is called as an expression vector.
These vectors are frequently engineered to contain regulatory
sequences that act as promoter and/or enhancer regions and lead
to efficient transcription of the insert gene.
Expression vectors are used for molecular biology techniques such
as site-directed mutagenesis.
 The goal of a well-designed expression vector is the production of
large amounts of stable messenger RNA, and in extension,
proteins.
Expression vectors are basic tools for biotechnology and the
production of proteins.
EXPRESSION VECTOR
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Strong promoter
Intact ORF
Ribosomal binding site
Termination sequence
CHOICE OF EXPRESSION VECTORS
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Goal Component
Insert cargo into the plasmid
and verify the insert sequence accuracy
MCS – restriction sites OR recombination
regions 5’ and 3’ Primer sites for sequence
verification
Insert plasmid into cells,
enable the plasmid to replicate inside the
host, & select for cells carrying the plasmid
•Backbone compatible with cloning method
• Origin of replication
• Selection marker and/or screening marker
Transcribe mRNA from the plasmid •Promoter (constitutive or inducible)
operator, terminator
Translate mRNA into protein Ribosome Binding Site, start codon, stop
codon
Promote proper folding of nascent protein •co-expression of chaperones
•Solubilization tags
•custom-designed synthetic RBS
•Codon-optimized ORF
Detect or Purify target protein •Epitope tags (His)
•reporters (GFP)
EXPRESSION VECTOR COMPONENTS
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1. PROKARYOTIC EXPRESSION VECTOR
 Bacterial expression system (e.g. E.coli)
2. EUKARYOTIC EXPRESSION VECTOR
 Yeast expression system (e.g. S.cervesiae)
 Viral expression system (e.g. Baculovirus)
 Mammalian cell expression system
EXPRESSION VECTOR TWO TYPE
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Host Commonly used methods to introduce expression
construct (plasmid)
Prokaryotic cells Transformation via • CaCl2 + heatshock
• electroporation
Yeast cells Transformation via • LiAc/PEG/ssDNA
• Electroporation
• spheroplasts, biolistics, glass
beads
Plant cells Transformation via • Agrobacterium-mediated
transformation
• Gene gun
Mammalian cell lines Transfection via •Liposomes
•Electroporation
•calcium phosphate, nanoparticles
Mammalian (primary) cells Transduction via lentivirus
in vivo delivery into live
animals
Transduction via • adenovirus
• AAV
DELIVERY METHODS
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SELECTION / SCREENING MARKERS
Host Selection Markers
Prokaryotic cells Ampicillin, Kanamycin, tetracycline,
chloramphenicol
Mammalian cells Neomycin; Puromycin; Hygromycin;
Zeocin
Yeast HIS4, Zeocin
Auxotrophy: URA3, TRP1, LEU2,
HIS3
Host Screening Markers
Prokaryotic cells Lacz for Blue-white screening
Prokaryotic or Eukaryotic cells Fluorescent Proteins (GFP, mCherry)
Luciferase
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TRANSCRIPTIONAL PROMOTERS
Host Commonly Used Promoters
Bacteria •Lac
•T7
•araBAD
Yeast •GAL4
•PGK
• ADH1
•ADE2
•TRP1
Mammalian •Constitutive: CMV, SV40, EF1a,
CAG
•Inducible: Tet
•Tissue-specific for in vivo work:
varied
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EPITOPE TAGS / FUSION PROTEINS
Goal Commonly Use Tag
Detect target protein (common Ab
epitope tags)
FLAG (DYKDDDK)
HA (YPYDVPDYA)
Purify target protein (affinity tags) His6
glutathione-S-transferase (GST)
Improve solubility of target protein maltose-binding protein (MBP)
glutathione-S-transferase (GST)
Tags can be removed at cleavage sites place between target ORF and tag:
Enzyme Cleavage site
Thrombin Leu-Val-Pro-Arg-Gly-Ser
Enterokinase Asp-Asp-Asp-Asp-Lys
Factor Xa Ile-Glu/Asp-Gly-Arg
TEV Glu-Asn-Leu-Tyr-Phe-Gln-Gly9

Saccharomyces cerevisiae
It is the most common eukaryotic system and there is a great deal of
study about this organism
It is a single-celled and behaves like a bacterial culture and can be
grown in relatively simple media in both small and large-scale
production
Well characterized with many strong regulatory promoters with
naturally occurring plasmids
Carry out post-translational modifications
Secretes very few of its own proteins
Recognized as safe by USDA and FDA
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Vaccines Diagnostics Human therapeutic
agents
•Hepatitis B virus
surface antigen
•Malaria
circumsporozine
protein
•HIV-1 envelope
protein
•Hepatitis C virus
protein
•HIV-1 antigens
•Epidermal growth
factor
•Insulin
•Fibroblast growth
factor
•Hirudin
•Human growth factor
Recombinant proteins produced by S. cerevisiae
expression systems
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There are three main classes of S. cerevisiae expression vectors.
• Yeast episomal plasmids (YEps)
• Yeast integrating plasmids (YIps)
• Yeast artificial chromosomes (YACs)
 Yeast episomal plasmids have been used extensively for the
production of either intra- or extracellular heterologous proteins
Typically, vectors function in both E. coli and S.cerevisiae.
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The YEps vectors are based on the high-copy-number 2μm plasmids
The vectors replicate independently via a single origin of replication.
There are more than 30 copies per cell.
Selection scheme rely on mutant host strains that require a particular
amino acid (histidine, tryptophan, or leucine) or nucleotide (uracil).
When a Yep with a wild-type LEU2 gene is transformed into a mutant
leu2 host cell, only cells that carry plasmid will grow.
A Yip vector is used to integrate a heterologous gene into the host
genome to provide a more reliable production system.
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Integration of DNA with a Yip vector
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YAC CLONING SYSTEM
A YAC is designed to clone a large segment of DNA
(100kb), which is then maintained as a separate chromosome in
the host yeast cell.
It is highly stable and has been used for the physical mapping
of human genomic DNA, the analysis of transcription units,
and genomic libraries.
It has a sequences that act as ARS for replication,
centromere for cell division, and telomere for stability.
To date, they have not been used as expression systems for
the commercial production.
15

Pichia pastoris Expression Systems
Though S. cerevisae is successfully used to produce
recombinant proteins for human, it has major drawbacks.
The level of protein production is low.
There is the tendency for hyperglycosylation resulting in
change of protein function.
Proteins are often retained in periplasm, increasing time
and cost for purification.
It produces ethanol at high cell densities, which is toxic
to cells.
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P. pastoris is a methylotrophic yeast that is able to utilize
methanol as a source of carbon and energy.
Glycosylation occurs to a lesser extent and the linkages
between sugar residues are of the α-1,2 type.
P. pastoris strain was extensively engineered with the aim
of developing a “humanized” strain that glycosylate proteins
in a manner identical to that of human cells.
It does not produce ethanol.
It normally secretes very few proteins, thus simplifying
the purification of secreted recombinant proteins.
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A double recombination event between the AOX1p and
AOX1 regions of the vector and the homologous segments of
chromosome DNA results in the insertion of the DNA
carrying the gene of interest and the HIS4 gene.
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Baculovirus-Insect Cell Expression
Baculoviruses are a large, diverse group of viruses that
specifically infect arthropods, and are not infectious to
other animals.
During the infection cycle, two forms of baculovirus
are produced.
A single nucleocaspid (virus particle) which can infect
more midgut cells.
Clusters of nucleocaspids that are produced outside of
the cells (virions) in a protein matrix (polyhedrin).
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The polyhedrin gene is replaced with a coding sequence
for a heterologous protein, followed by infection of cultured
insect cells, resulting in the production of the heterologous
protein.
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 Constructs have been made using the polyhedrin
promoter to produce large quantities of extracellular
protein.
Most proteins are modified and secreted properly.
Grows very well in many insect cell lines allowing easy
production.
Minor problem that (galactose and sialic acid; N-linked.)
doesn’t process certain mammalian glycosylation types
correctly
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• The specific baculovirus that has been used extensively is
Autographa californica multiple nuclear polyhedrosis virus
(AcMNPV.)
• A gene of interest is inserted into the MCS and the transfer
vector is propagated in E. coli.
• Next, insect cells in culture are cotransfected with AcMNPV
DNA and the transfer vector carrying the cloned gene.
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Mammalian Cell Expression Systems
Important for producing proteins with all post
translational modifications.
Many established cell lines are useful.
Transient expression: African green monkey, baby
hamster & human embryonic (all kidney tissue cell lines.)
Long-term expression: Chinese hamster ovary and
mouse myeloma cells.
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Expression vectors in these systems are usually derived
from an animal virus such as SV40 (simian virus 40).
Can be used for expression of single polypeptides,
homooligomers, and heterooligomers.
The latter is made possible by transforming with two or
more separate cloned genes.
Industrial production is however costly.
Mammalian Cell Expression Systems
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VECTOR DESIGN
Generalized mammalian expression vector.
The MCS and SMG (Selectable Marker Gene) are under the
control of eukaryotic promoter (p), polyadenylation(pa),
and terminal sequence(TT).
An intron(I) enhances the production of heterologous
protein.
The Ampr gene is used for selecting transformed E. coli.
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For the best results, a gene of interest must be equipped with
translation control sequences.
A gene of interest can be fitted with various sequences that
enhance translation and facilitate both secretion and
purification.
A Kozak sequence(K), specific sequence surrounding the
AUG start codon, signal sequence(S), protein affinity tag(T)
for purification, proteolytic cleavage site(P), and stop
codon(SC).
The 5’ and 3’ UTR increase the efficiency of translation and
contribute to mRNA stability.
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Two-Vector Expression System
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Two-Vector Expression System
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Baculovirus Vector in Mammalian Cells
It is possible to use some of the baculovirus vector to express
target proteins in mammalian cells.
Because baculovirus cannot replicate in mammalian cells
and the polyhedron-deficient strains employed as vectors
cannot infect insects.
It is a safe system.
For stable long-term expression, the target gene is inserted
between sequences for adeno-associated virus inverted
terminal repeat to facilitate the integration into the host cells.
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pBAD
 The pBAD/His and pBAD/Myc-His plasmids are pBR322-derived expression
vectors designed for regulated, dose-dependent recombinant protein expression and
purification in E. coli.
 Optimum levels of soluble, recombinant protein are possible using the araBAD
promoter (PBAD) from E. coli.
 The regulatory protein, AraC, is provided on the pBAD/His and pBAD/Myc-His
vectors allowing regulation of PBAD.
 In the presence of L-arabinose, expression from PBAD is turned on while the absence
of L-arabinose produces very low levels of transcription from PBAD (Lee, 1980; Lee et
al., 1987).
 By varying the concentration of L-arabinose, protein expression levels can be
optimized to ensure maximum expression of soluble protein.
 In addition, the tight regulation of PBAD by AraC is useful for expression of
potentially toxic or essential genes (Carson et al., 1991; Dalbey and Wickner, 1985;
Guzman et al., 1992; Kuhn and Wickner, 1985; Russell et al., 1989; San Millan et al.,

The construction of expression libraries.
The analysis of gene function at protein level.
The commercial production of proteins.
The production of antibodies.
For in vivo studies of the protein.
APPLICATION OF EXPRESSION VECTOR
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REFERENCES
 Expression vectors: how to choose or customize
vectors for gene & protein expression - Rachel
Speer, Ph.D. Molecular Biology Specialist
 Expresion system BY C.SWORNA KUMARI
M.Sc.,M.PHIL BIOTECHNOLOGY
 Invitrogen Life Technologies
pBAD/His A, B, and C
pBAD/Myc-His A, B, and C
Vectors for Dose-Dependent Expression of
Recombinant Proteins Containing N- or C-
Terminal 6xHis Tags in E. coli
Catalog nos. V430-01, V440-01
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Exprssion vector

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