This presentation covers a general introduction to expression vector, its components, types, and its application. Then it covers some of the expression system with examples.
2. 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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4. 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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5. 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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6. 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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8. 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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9. 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
10. 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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11. 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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12. 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.
Saccharomyces cerevisiae
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13. 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.
Saccharomyces cerevisiae
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15. 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.
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17. 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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18. 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.
Pichia pastoris Expression Systems
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19. 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.
Pichia pastoris Expression Systems
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21. 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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22. 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.
Baculovirus-Insect Cell Expression
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23. 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
Baculovirus-Insect Cell Expression
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24. • 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.
Baculovirus-Insect Cell Expression
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26. 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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27. 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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28. 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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29. 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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32. 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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33. 33
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.,
36. 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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37. 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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Site-directed mutagenesis is a molecular biology method that is used to make specific and intentional changes to the DNA sequence of a gene and any gene products. Also called site-specific mutagenesis or oligonucleotide-directed mutagenesis, it is used for investigating the structure and biological activity of DNA, RNA, and protein molecules, and for protein engineering.
Site-directed mutagenesis is one of the most important techniques in laboratory for introducing a mutation into a DNA sequence. There are numerous methods for achieving site-directed mutagenesis, but with decreasing costs of oligonucleotide synthesis, artificial gene synthesis is now occasionally used as an alternative to site-directed mutagenesis. Since 2013, the development of the CRISPR/Cas9 technology, based on a prokaryotic viral defense system, has also allowed for the editing of the genome, and mutagenesis may be performed in vivo with relative ease
In molecular genetics, an open reading frame(ORF) is the part of a reading frame that has the ability to be translated. An ORF is a continuous stretch of codons that contain a start codon (usually AUG) and a stop codon (usually UAA, UAG or UGA).
Recombination hotspots are regions in a genome that exhibit elevated rates of recombination relative to a neutral expectation. The recombination rate within hotspots can be hundreds of times that of the surrounding region.[1] Recombination hotspots result from higher DNA break formation in these regions, and apply to both mitotic and meiotic cells. This appellation can refer to recombination events resulting from the uneven distribution of programmed meiotic double-strand breaks
The most basic type of promoter you can have in a prokaryote are constitutive promoters. These promoters initiate mRNA synthesis independent of the influence of regulation. The term 'constititive expression' is more commonly defined as 'Expression of a gene that is transcribed at a constant level'.
Inducible promoters are a very powerful tool in genetic engineering because the expression of genes operably linked to them can be turned on or off at certain stages of development of an organism or in a particular tissue
In molecular biology, molecular chaperones are proteins that assist the covalent folding or unfolding and the assembly or disassembly of other macromolecular structures.
transformation is the genetic alteration of a cell resulting from the direct uptake and incorporation of exogenous genetic material from its surroundings through the cell membrane
transduction (injection of foreign DNA by a bacteriophage virus into the host bacterium)
Transformation" may also be used to describe the insertion of new genetic material into nonbacterial cells, including animal and plant cells; however, because "transformation" has a special meaning in relation to animal cells, indicating progression to a cancerous state, the process is usually called "transfection
Lithium acetate is also used to permeabilize the cell wall of yeast for use in DNA transformation. It is believed that the beneficial effect of LiOAc is caused by its chaotropic effect; denaturing DNA, RNA and proteins
Calcium chloride (CaCl2) transformation is a laboratory technique in prokaryotic (bacterial) cell biology. It increases the ability of a prokaryotic cell to incorporate plasmid DNAallowing them to be genetically transformed.[1] The addition of calcium chloride to a cell suspension promotes the binding of plasmid DNA to lipopolysaccharides (LPS). Positively charged calcium ions attract both the negatively charged DNA backbone and the negatively charged groups in the LPS inner core. The plasmid DNA can then pass into the cell upon heat shock, where chilled cells (+4 degrees Celsius) are heated to a higher temperature (+42 degrees Celsius) for a short time.
The blue–white screen is a screening technique that allows for the rapid and convenient detection of recombinant bacteria in vector-based molecular cloning experiments. DNA of interest is ligated into a vector. The vector is then inserted into a competent host cellviable for transformation, which are then grown in the presence of X-gal. Cells transformed with vectors containing recombinant DNA will produce white colonies; cells transformed with non-recombinant plasmids (i.e. only the vector) grow into blue colonies. This method of screening is usually performed using a suitable bacterial strain, but other organisms such as yeast may also be used
Two of the most important agencies that primarily affect our food every single day are the United States Department of Agriculture (USDA) and the Food and Drug Administration (FDA)
The word “episomal” indicates that a YEp can replicate as an independent plasmid, but also implies that integration into one of the yeast chromosomes can occur. Integration occurs because the gene carried on the vector as a selectable marker is very similar to the mutant version of the gene present in the yeast chromosomal DNA
Yeast Integrating plasmids (YIp): These plasmids lack an ORI and must be integrated directly into the host chromosome via homologous recombination. Yeast Replicating plasmids (YRp): These vectors contain an Autonomously Replicating Sequence (ARS) derived from the yeast chromosome
Yeast artificial chromosomes (YACs) are genetically engineered chromosomes derived from the DNA of the yeast, Saccharomyces cerevisiae, which is then ligated into a bacterial plasmid
Autonomously replicating sequence. An autonomously replicating sequence(ARS) contains the origin of replication in the yeast genome. It contains four regions (A, B1, B2, and B3), named in order of their effect on plasmid stability. The A-Domain is highly conserved, any mutation abolishes origin function.
URA3 is often used in yeast research as a "marker gene", that is, a gene to label chromosomes or plasmids
Thermo Scientific SmaI restriction enzyme recognizes CCC^GGG sites and cuts best at 30°C in Tango buffer
the process of adding glycosyl groups to a protein to form a glycoprotein.
P. pastoris has two alcohol oxidase genes, Aox1 and Aox2, which have a strongly inducible promoter.[2] These genes allow Pichia to use methanol as a carbon and energy source. The AOX promoters are induced by methanol and are repressed by e.g. glucose. Usually, the gene for the desired protein is introduced under the control of the Aox1 promoter, which means that protein production can be induced by the addition of methanol
The baculovirus life cycle involves two distinct forms of virus. Occlusion derived virus (ODV) is present in a protein matrix (polyhedrin or granulin) and is responsible for the primary infection of the host while the budded virus (BV) is released from the infected host cells later during the secondary infection.
Transient expression is the temporary expression of genes that later in development are no longer expressed. The transient expression is a fundamental technique of gene expression in hosts of interest for a limited time.
SV40 is an abbreviation for simian vacuolating virus 40 or simian virus 40, a polyomavirus that is found in both monkeys and humans. It was named for the effect it produced on infected green monkey cells, which developed an unusual number of vacuoles. Like other polyomaviruses, SV40 is a DNA virus that has the potential to cause tumors in animals, but most often persists as a latent infection. SV40 has been widely studied as a model eukaryotic virus, leading to many early discoveries in eukaryotic DNA replication[1] and transcription.
In molecular genetics, an untranslated region (or UTR) refers to either of two sections, one on each side of a coding sequence on a strand of mRNA. If it is found on the 5' side, it is called the 5' UTR (or leader sequence), or if it is found on the 3' side, it is called the 3' UTR (or trailer sequence). mRNA is RNA that carries information from DNA to the ribosome, the site of protein synthesis (translation) within a cell. The mRNA is initially transcribed from the corresponding DNA sequence and then translated into protein. However, several regions of the mRNA are usually not translated into protein, including the 5' and 3' UTRs
inverted terminal repeats (ITR) that aid in concatemer formation in the nucleus after the single-stranded vector DNA is converted by host cell DNA polymerase complexes into double-stranded DNA