Cloning of the Gene, Purification as Recombinant Protein ...
Expression and purification of recombinant proteins in Bacterial and yeast system
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Transcript of Expression and purification of recombinant proteins in Bacterial and yeast system
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Expression and purification of recombinant
proteins in E.coli and Yeast system
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Steps to produce recombinant protein
1.Amplification of gene of interest.
2.Insert into cloning vector.
3.Sub cloning into expression vector.
4.Transformation into protein expressing bacteria (E coli) or yeast.
5.Test for identification of recombinant protein.
6.Large scale production.
7.purification.
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Factors
1.Which expression vector ?
2.Which host system?
3.Properties of protein
•Membrane bound
•Solubility
•Single or multidomain
•Size
4.Where it expressed?
5. purification method?
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Expression Choices
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Features of expression vector
•Origin of replication
•Drug resistance marker
•A promoter
•Transcription terminator
•Restriction sites
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Expression Vectors for E.coli
pET
Strong expression of GOI – up to 50%
of total cell protein.
Uses strong promoter of T7 RNA
polymerase
strong control of expression
not leaky
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pBAD
Induction by addition of arabinose
Sugar binds to Ara protein
Complex binds and initiates RNA polymerase
Good choice for toxic proteins
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pQE –
Low copy plasmid with a T5 promoter
Two lac operon sequence for repressor binding
These can be leaky promoters
IPTG inducable
pGEX–
IPTG inducable
Ptac promoter
Fusion protein – Glutathione S Transferase (GST)
Easy to purify
Good expression for fusion proteins
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Popular promoters for heterologous protein
expression in E. coli
1. Plac
2. Ptrp
3. Hybrid promoters
4. pBAD
5. T7
6. T5
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Where to express the recombinant proteins?
1. Direct expression (cytosol):
difficult to ensure proper di-sulphide bonds formation.
2. Fusion expression :
Ensures good translation initiation. Can overcome insolubility problems with small peptides.
3. Secretion (periplasm or medium):
Periplasm offers a more oxidizing environment, where proteins tend to fold better.
inability for posttranslational modifications of proteins.
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Inclusion bodies
Region of bacteria which can fill with insoluble protein
Over expression of proteins or toxic proteins induce formation of inclusion bodies
Aggregates may be mostly the expressed protein
disulfide bonds incorrectly formed
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Pros and Cons of E.coli system
Advantages
High yield
easier scale up
inexpensive media
Many expression plasmid
Fast growth
Disadvantages
No post-translational modification
Large proteins may be difficult to
express
Membrane proteins may not fold
well
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WHY IS YEAST PREFERRED ?
Can be cultured easily in small vessels or large bioreactors.
Well known genetically & physiologically.
Can be easily manipulated.
Several promoters isolated.
Capable of post -translational modifications.
Product can be readily purified.
Recognized safe (GRAS) organism by US Food & Drug Administration.
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YEAST VECTORS
There are 3 types of yeast expression vectors .
o Episomal or plasmid vectors (YEps)
o Integrating vectors (YIps)
o Yeast artificial chromosomes (YACs)
A typical yeast vector consists of : Ori , Promoter,
Selection marker, Terminator & Polylinker.
Yeps
extensively used
high copy number.
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YAC vector
YACs in addition have Centromeric & Telomeric sequences which
are host specific.
ARS : autonomously replicating sequences (ORI)
YIp : Yeast integrative plasmids
URA gene facilitates growth of yeast on media not supplemented
With uracil.
Genetic marker for DNA transformations.
YRp
Yeast replicative plasmid
Tryptophan marker
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Yeast Expression strains
S. cerevisae
Kluyveromyces lactis
Pichia pastoris
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PROBLEMS ASSOCIATED WITH S. cerevisae
Low expression & modest yield.
Proteins are often hyperglycosylated.
Excess mannose residues alters the function
Sometimes proteins are retained in the periplasmic space & this
increases the cost of purification.
It also produces ethanol at high cell densities, which is toxic to the
cells.
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Pros and Cons of yeast system
Advantages
•Lacks detectable endotoxins.
•Fermentation relatively inexpensive.
•Facilitates glycosylation and
formation of disulphide bonds.
•Only 0.5% native proteins are
secreted so isolation of secreted
product is simplified.
Disadvantages
•Gene expression less easily
controlled.
•Glycosylation not identical to
mammalian systems.
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Various purification methods:
1.Charge: IEC/IEF
2.Size: size exclusion chromatography
3.Hydrophobicity: Hydrophobic Interaction Chromatography
4. affinity chromatography
5.Solubility: Precipitation
6. Dialysis
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SDS-PAGE
purification methods
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Gel filtration chromatography - separation by size
Beads have different size pores
As column flows:
• large proteins excluded from
pores
and therefore flow rapidly
• small proteins enter pores and
flow slowly
Resins: polyacrylamide, agarose,
dextrin
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Ion exchange chromatography – separation by charge
Beads have charged group: + charge binds acidic amino acids- charge binds basic amino acid
Different proteins bind with different affinity
Eluted with increasing amount of salt (NaCl or KCl)
Different proteins elute at different salt concentrations
Anion resin :diethylaminoethyl
(DEAE)
Cation : carboxymethyl
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Affinity chromatography
separation by biological binding interactions
wash
porous
bead elute
apply sample
protein of interest
Beads: Cellulose
agarose
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Increase selectivity of
protein purification:
(Gene fusion strategies)
Most target protein lack a suitable
Affinity ligand usable for capture on
a solid matrix. A way to circumvent this
obstacle is to genetically fuse the gene
encoding the target protein with a gene
encoding a purification tag. When the
chimeric protein is expressed, the tag
allows for specific capture of the fusion
protein. This will allow the purification
of virtually any protein without any prior
knowledge of its biochemical properties.
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Tags
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His tags His tags are typically a series of 6 histidines added to the C or N terminus of a
recombinant protein
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Resin (IMAC nickel)
Ni
His-tagged
Recombinant
Protein
• His tag and column interaction
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His tags
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Imidazole
N3H+-OOC
Histidine
• His and imidazole structure similarities
• Imidazole competes with His for Ni2+ sites
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GST Tag
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Strep Tag
PBS buffer (wash)Removal
indicated by
Red to yellow
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FLAG Tag
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T7 Tag
11aa T7 Tag sequence
binding target proteins to T7
Tag y which is covalently
coupled to cross-linked agarose
beads
Wash and elute
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Advantages and disadvantages for
using tags in fusion proteins
Advantages
(1)improve protein yield
(2) prevent proteolysis
(3) facilitate protein refolding
(4) protect the antigenicity of the fusion protein
(5) increase solubility
(6) increased sensitivity
Disadvantages
(1) a change in protein conformation
(2) lower protein yields
(3) inhibition of enzyme activity
(4) alteration in biological activity
(5) undesired flexibility in structural studies
(6) cleavage/removing the fusion partner
requires expensive protease
(7) toxicity.
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HUMAN THERAPEUTIC AGENTS
Epidermal growth factor
Insulin
Insulin- like growth factor
Platelet- derived growth factor
Proinsulin
Fibroblast growth factor
Granulocyte- macrophage colony stimulating factor
Antitrypsin
Blood coagulation factor XIIIa
Hirudin
Human growth factor
Human serum albumin
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Applications
Functional Studies
Enzymatic Assays
Protein-protein interactions
Protein Ligand Interactions
Structural Studies
Protein Crystallography & NMR Structure Determination
Target Proteins for Rational Drug Design
Therapeutic Proteins – Preclinical Studies
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