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Expression and purification of recombinant
proteins in E.coli and Yeast system
Steps to produce recombinant protein
1.Amplification of gene of interest.
2.Insert into cloning vector.
3.Sub cloning into...
Factors
1.Which expression vector ?
2.Which host system?
3.Properties of protein
•Membrane bound
•Solubility
•Single or mu...
Expression Choices
Features of expression vector
•Origin of replication
•Drug resistance marker
•A promoter
•Transcription terminator
•Restri...
Expression Vectors for E.coli
pET
 Strong expression of GOI – up to 50%
of total cell protein.
 Uses strong promoter of ...
pBAD
 Induction by addition of arabinose
 Sugar binds to Ara protein
 Complex binds and initiates RNA polymerase
 Good...
pQE –
 Low copy plasmid with a T5 promoter
 Two lac operon sequence for repressor binding
 These can be leaky promoters...
Popular promoters for heterologous protein
expression in E. coli
1. Plac
2. Ptrp
3. Hybrid promoters
4. pBAD
5. T7
6. T5
Where to express the recombinant proteins?
1. Direct expression (cytosol):
difficult to ensure proper di-sulphide bonds fo...
Inclusion bodies
 Region of bacteria which can fill with insoluble protein
 Over expression of proteins or toxic protein...
Pros and Cons of E.coli system
Advantages
 High yield
 easier scale up
 inexpensive media
 Many expression plasmid
 F...
WHY IS YEAST PREFERRED ?
 Can be cultured easily in small vessels or large bioreactors.
 Well known genetically & physio...
YEAST VECTORS
 There are 3 types of yeast expression vectors .
o Episomal or plasmid vectors (YEps)
o Integrating vectors...
YAC vector
 YACs in addition have Centromeric & Telomeric sequences which
are host specific.
 ARS : autonomously replica...
Yeast Expression strains
 S. cerevisae
Kluyveromyces lactis
Pichia pastoris
PROBLEMS ASSOCIATED WITH S. cerevisae
 Low expression & modest yield.
 Proteins are often hyperglycosylated.
 Excess ma...
Pros and Cons of yeast system
Advantages
•Lacks detectable endotoxins.
•Fermentation relatively inexpensive.
•Facilitates ...
Various purification methods:
1.Charge: IEC/IEF
2.Size: size exclusion chromatography
3.Hydrophobicity: Hydrophobic Intera...
SDS-PAGE
purification methods
Gel filtration chromatography - separation by size
Beads have different size pores
As column flows:
• large proteins exclu...
Ion exchange chromatography – separation by charge
Beads have charged group:
+ charge binds acidic amino acids
- charge bi...
Affinity chromatography
separation by biological binding interactions
wash
porous
bead elute
apply sample
protein of inter...
Increase selectivity of
protein purification:
(Gene fusion strategies)
Most target protein lack a suitable
Affinity ligand...
Tags
His tags
 His tags are typically a series of 6 histidines added to the C or N terminus of a
recombinant protein
27
Resin ...
His tags
28
Imidazole
N3H+-OOC
Histidine
• His and imidazole structure similarities
• Imidazole competes with His for Ni2+...
GST Tag
Strep Tag
PBS buffer (wash)Removal
indicated by
Red to yellow
FLAG Tag
T7 Tag
 11aa T7 Tag sequence
 binding target proteins to T7
Tag y which is covalently
coupled to cross-linked agarose
be...
Advantages and disadvantages for
using tags in fusion proteins
Advantages
(1)improve protein yield
(2) prevent proteolysis...
HUMAN THERAPEUTIC AGENTS
 Epidermal growth factor
 Insulin
 Insulin- like growth factor
 Platelet- derived growth fact...
Applications
Functional Studies
Enzymatic Assays
Protein-protein interactions
Protein Ligand Interactions
Structural ...
Expression and purification of recombinant proteins in Bacterial and yeast system
Expression and purification of recombinant proteins in Bacterial and yeast system
Expression and purification of recombinant proteins in Bacterial and yeast system
Expression and purification of recombinant proteins in Bacterial and yeast system
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Expression and purification of recombinant proteins in Bacterial and yeast system

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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.

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Expression and purification of recombinant proteins in Bacterial and yeast system

  1. 1. Expression and purification of recombinant proteins in E.coli and Yeast system
  2. 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. 3. 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?
  4. 4. Expression Choices
  5. 5. Features of expression vector •Origin of replication •Drug resistance marker •A promoter •Transcription terminator •Restriction sites
  6. 6. 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
  7. 7. pBAD  Induction by addition of arabinose  Sugar binds to Ara protein  Complex binds and initiates RNA polymerase  Good choice for toxic proteins
  8. 8. 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
  9. 9. Popular promoters for heterologous protein expression in E. coli 1. Plac 2. Ptrp 3. Hybrid promoters 4. pBAD 5. T7 6. T5
  10. 10. 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.
  11. 11. 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
  12. 12. 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
  13. 13. 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.
  14. 14. 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.
  15. 15. 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
  16. 16. Yeast Expression strains  S. cerevisae Kluyveromyces lactis Pichia pastoris
  17. 17. 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.
  18. 18. 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.
  19. 19. 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
  20. 20. SDS-PAGE purification methods
  21. 21. 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
  22. 22. 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
  23. 23. Affinity chromatography separation by biological binding interactions wash porous bead elute apply sample protein of interest Beads: Cellulose agarose
  24. 24. 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.
  25. 25. Tags
  26. 26. His tags  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 • His tag and column interaction
  27. 27. His tags 28 Imidazole N3H+-OOC Histidine • His and imidazole structure similarities • Imidazole competes with His for Ni2+ sites
  28. 28. GST Tag
  29. 29. Strep Tag PBS buffer (wash)Removal indicated by Red to yellow
  30. 30. FLAG Tag
  31. 31. 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
  32. 32. 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.
  33. 33. 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
  34. 34. 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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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.

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