Expression system final

Expresion system
BY
C.SWORNA KUMARI
M.Sc.,M.PHIL BIOTECHNOLOGY

expression vector
 Definition :
An expression vector, otherwise
known as an expression construct, is
usually a plasmid or virus designed
for protein expression in cells

Difference between cloning vectors
and expression vectors
 Cloning vectors:
 Cloning vectors are the DNA molecules that can carry a foreign dna
segment in to the host cell.The vectors usd in rDNA technology can be
 Plasmids:Self replicating,circular,extra chromosal dna present in
bacteria.plasmids have one or two copies per cell
 Bacteriophages:Virus infecting bacteria.bacteriophages have a high
copy number per cell, so their copy number is also high in genome
 Cosmids:Hybrid vectors derived from plasmids which contain cos site
of lambda phage

Expression vectors
 The cloning vector containing suitable expression signals to have
maximum gene for expression is called expression vector.
 The following expression signals are introduced in to gene cloned
vectors to get maximum expression:
 Insertion of a strong promoter.
 Insertion of a strong termination codon.
 Adjustment of distance between promoter and cloned gene.
 Insertion of transcription termination sequence.
 Insertion of a strong translation initiation sequence.

. Promoter –commonly used inducible promoters are
promoters derived from lac operon and the T7 promoter.
Other strong promoters used include Trp
promoter and Tac Promoter, which a hybrid of both the
Trp and Lac Operon promoters
Ribosome binding site (RBS) Follows the promoter,
and promotes efficient translation of the protein of
interest.
Translation initiation site:shine dalgarno sequence
enclosed in the RBS,8 base-pairs upstream of the AUG
strat codon
Expression in Prokaryotes

• Eukaryotic protein synthesis occurs in cytoplasm or on
the endoplasmic reticulum.
• These proteins are further post translational processed
that is required for protein activity and stability.
• Disulfide isomerase also makes sure that the proteins
produced have the correct configuration.
• The proper glycosylation that are necessary for protein
conformation, localization by interacting with specific
receptor and increase stability.
Posttranslational Modification

Eukaryotic Expression
Systems
 Eukayrote expression vectors require sequences that encode for:
 Polyadenylation tail: Creates a polyadenylation tail at the end of the
transcribed pre-mRNA that protects the mRNA fromexonucleases and
ensures transcriptional and translational termination: stabilizes mRNA
production.
 Minimal UTR length:UTRs contain specific characteristics that may
impede transcription or translation, and thus the shortest UTRs or none
at all are encoded for in optimal expression vectors.
 Kozak sequence: Vectors should encode for a Kozak sequence in the
mRNA, which assembles the ribosome for translation of the mRNA

• The major features of a eukaryotic expression vector are a
promoter, a multiple cloning site, DNA segment for
termination and polyadenylation, selectable marker, origin of
replication in E. coli and eukaryotic cell and Ampr for
marker in E. coli.

Different types of expression
system:
 Prokaryotic expression systems yeast
 Escherichia coli Pichia pastoris
 Lactococcus lactis Pichia methanolica
 Other bacteria, e.g. Bacillus species Saccharomyces cerevisiae
 Insect cells mammalian cells
 Baculovirus Viral infection,
 Stable recombinant cell lines e.g adeno virus,reteo virus ,lenti virus etc
 Schneider cells (Drosophila)
 In vitro expression systems others
 Rabbit reticulocyte lysate (red blood cells) Xenopus oocytes and cell-free extract
 Wheat germ extract Transgenic mice,plants
 Escherichia coli extract Milk of transgenic animals

• Saccharomyces cerevisiae
• Pichia pastoris
• Baculovirus-insect cell lines
• Mammalian systems
Eukaryotic Expression Systems

• It is the most common eukaryotic system and there is a
great deal of study about this organism.
• It is a ingle-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 regulatable
promoters with naturally occurring plasmids.
• Carry out post-translational modifications.
• Secretes very few of its own proteins.
• Recognized as safe by USDA and FDA.
Saccharomyces cerevisiae

• 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 eitehr intra- or extracellular
heterologous proteins.
• Typically, vectors function in both E. coli and S.
cerevisiae.

• 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.

• Generally, tightly regulatable, inducible promoters are
preferred for producing large amounts of recombinant
protein at a specific time during large-scale growth.

• Most heterologous genes are provided with a DNA
coding sequence for signal peptide that facilitates the
secretion of protein through cell membranes and
external environment.
• Other sequence that protect the recombinant protein
from proteolytic degradation, and provide a affinity tag
is also used.
• These extra amino acid sequences are equipped with a
protease cleavage site so that they can be removed
from the recombinant protein.

• Plasmid-based yeast expression systems are often
unstable under large-scale growth conditions even in the
presence of selection pressure.
• A Yip vector is used to integrate a heterologous gene
into the host genome to provide a more reliable
production system.
• The plasmid does not usually carry an origin of
replication.
• The disadvantage is the low yield of recombinant
protein from a single gene copy.

Integration of DNA with a Yip vector

• A YAC is designed to clone a large segment of DNA (100
kb), 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.
YAC cloning system

• Human Cu/Zn SOD cDNA was cloned between the promoter
and termination-polyadenylation sequence of the yeast
GAPD gene and subsequently used to transform LEU-
mutant host cell.
Intercellular Production in Yeast

• Proteins may also be produced for secretion.
• In this system, any glycosylated protein is secreted (O or N-
linked).
• The coding sequences of recombinant proteins must be
cloned downstream of a leader sequence, the yeast mating
type factor α-factor.
• Under these conditions, correct disulfide bond formation,
proteolytic removal of the leader sequence, and appropriate
posttranslational modifications occur, and an active
recombinant protein is secreted.
• The leader peptide is removed by endoprotease that
recognizes the Lys-Arg.
Secretion of Heterologous Proteins

• For example, a properly processed and active form of the
protein hirudin; a powerful anticoagulant protein cloned from
a leech, was synthesized and secreted by an S. cerevisiae.
• A YEp vector that had the prepro-α-factor sequence added to
the huridin coding sequencea to allow expression that is
cleaved away in processing.
• Leaves active hirudin which is secreted.
• Producing a recombinant protein for use in human
therapeutics in yeast rather than in bacteria is to ensure the
proper folding.

• 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.
Pichia pastoris Expression Systems

• 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.

• 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.

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).

• 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.

• 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 doesn’t process certain mammalian
glycosylation types correctly (galactose and sialic acid;
N-linked.)

Baculovirus Expression Vectors
• 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 Expression Vectors

Increasing the
Yield of
Recombinant
Baculovirus

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.

Mammalian Cell Expression Systems
• 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.

Vector Design
• Generalized mammalian expression vector.
• The MCS and SMG are under the control of eukaryotic
promoter, polyadenylation, and terminal sequence.
• An intron enhances the production of heterologous protein.
• The Ampr gene is used for selecting transformed E. coli.

• 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, specific sequence surrounding the AUG
start codon, signal sequence, protein affinity tag for
purification, proteolytic cleavage site, and stop codon.
• The 5’ and 3’ UTR increase the efficiency of translation and
contribute to mRNA stability.

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.

Selectable Markers for mammalian
Expression Vectors

Expression system final

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