This presentation gives the information about bacterial and yeast system as host for expressing recombinant proteins, suitable vectors, strains of host, Pros and cons of this system, different purification techniques and commercially available proteins produced so far by this system.
2. 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.
3. Factors
1.Which expression vector ?
2.Which host system?
3.Properties of protein
âĸMembrane bound
âĸSolubility
âĸSingle or multidomain
âĸSize
4.Where it expressed?
5. purification method?
6. Features of expression vector
âĸOrigin of replication
âĸDrug resistance marker
âĸA promoter
âĸTranscription terminator
âĸRestriction sites
7. Expression Vectors for E.coli
pET
ī Strong expression of GOI â up to 50%
of total cell protein.
ī Uses strong promoter of T7 RNA
polymerase
ī strong control of expression
ī not leaky
8. pBAD
ī Induction by addition of arabinose
ī Sugar binds to Ara protein
ī Complex binds and initiates RNA polymerase
ī Good choice for toxic proteins
9. pQE â
ī Low copy plasmid with a T5 promoter
ī Two lac operon sequence for repressor binding
ī These can be leaky promoters
ī IPTG inducable
pGEXâ
ī IPTG inducable
ī Ptac promoter
ī Fusion protein â Glutathione S Transferase (GST)
ī Easy to purify
ī Good expression for fusion proteins
10. Popular promoters for heterologous protein
expression in E. coli
1. Plac
2. Ptrp
3. Hybrid promoters
4. pBAD
5. T7
6. T5
11. 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.
12. Inclusion bodies
īĩ Region of bacteria which can fill with insoluble protein
īĩ Over expression of proteins or toxic proteins induce formation of inclusion bodies
īĩ Aggregates may be mostly the expressed protein
īĩ disulfide bonds incorrectly formed
13. Pros and Cons of E.coli system
Advantages
īĩ High yield
īĩ easier scale up
īĩ inexpensive media
īĩ Many expression plasmid
īĩ Fast growth
Disadvantages
īĩ No post-translational modification
īĩ Large proteins may be difficult to
express
īĩ Membrane proteins may not fold
well
14. WHY IS YEAST PREFERRED ?
īĩ Can be cultured easily in small vessels or large bioreactors.
īĩ Well known genetically & physiologically.
īĩ Can be easily manipulated.
īĩ Several promoters isolated.
īĩ Capable of post -translational modifications.
īĩ Product can be readily purified.
īĩ Recognized safe (GRAS) organism by US Food & Drug Administration.
15. YEAST VECTORS
īĩ There are 3 types of yeast expression vectors .
o Episomal or plasmid vectors (YEps)
o Integrating vectors (YIps)
o Yeast artificial chromosomes (YACs)
īĩ A typical yeast vector consists of : Ori , Promoter,
Selection marker, Terminator & Polylinker.
Yeps
īĩ extensively used
īĩ high copy number.
16. YAC vector
ī YACs in addition have Centromeric & Telomeric sequences which
are host specific.
ī ARS : autonomously replicating sequences (ORI)
YIp : Yeast integrative plasmids
ī URA gene facilitates growth of yeast on media not supplemented
With uracil.
ī Genetic marker for DNA transformations.
YRp
ī Yeast replicative plasmid
ī Tryptophan marker
18. PROBLEMS ASSOCIATED WITH S. cerevisae
īĩ Low expression & modest yield.
īĩ Proteins are often hyperglycosylated.
īĩ Excess mannose residues alters the function
īĩ Sometimes proteins are retained in the periplasmic space & this
increases the cost of purification.
īĩ It also produces ethanol at high cell densities, which is toxic to the
cells.
19. Pros and Cons of yeast system
Advantages
âĸLacks detectable endotoxins.
âĸFermentation relatively inexpensive.
âĸFacilitates glycosylation and
formation of disulphide bonds.
âĸOnly 0.5% native proteins are
secreted so isolation of secreted
product is simplified.
Disadvantages
âĸGene expression less easily
controlled.
âĸGlycosylation not identical to
mammalian systems.
22. Gel filtration chromatography - separation by size
Beads have different size pores
As column flows:
âĸ large proteins excluded from
pores
and therefore flow rapidly
âĸ small proteins enter pores and
flow slowly
Resins: polyacrylamide, agarose,
dextrin
23. Ion exchange chromatography â 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
24. Affinity chromatography
separation by biological binding interactions
wash
porous
bead elute
apply sample
protein of interest
Beads: Cellulose
agarose
25. 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.
27. His tags
īĩ His tags are typically a series of 6 histidines added to the C or N terminus of a
recombinant protein
27
Resin (IMAC nickel)
Ni
His-tagged
Recombinant
Protein
âĸ His tag and column interaction
33. T7 Tag
ī 11aa T7 Tag sequence
ī binding target proteins to T7
Tag y which is covalently
coupled to cross-linked agarose
beads
ī Wash and elute
34.
35. 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.
36. HUMAN THERAPEUTIC AGENTS
īĩ Epidermal growth factor
īĩ Insulin
īĩ Insulin- like growth factor
īĩ Platelet- derived growth factor
īĩ Proinsulin
īĩ Fibroblast growth factor
īĩ Granulocyte- macrophage colony stimulating factor
īĩ Antitrypsin
īĩ Blood coagulation factor XIIIa
īĩ Hirudin
īĩ Human growth factor
īĩ Human serum albumin