1.
Expression and purification of recombinant
proteins in E.coli and Yeast 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
•Membrane bound
•Solubility
•Single or multidomain
•Size
4.Where it expressed?
5. purification method?

4.
Expression Choices

5.
Features of expression vector
•Origin of replication
•Drug resistance marker
•A promoter
•Transcription terminator
•Restriction sites

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.
pBAD
 Induction by addition of arabinose
 Sugar binds to Ara protein
 Complex binds and initiates RNA polymerase
 Good choice for toxic proteins

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.
Popular promoters for heterologous protein
expression in E. coli
1. Plac
2. Ptrp
3. Hybrid promoters
4. pBAD
5. T7
6. T5

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.
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.
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.
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.
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.
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.
Yeast Expression strains
 S. cerevisae
Kluyveromyces lactis
Pichia pastoris

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.
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.
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.
SDS-PAGE
purification methods

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.
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.
Affinity chromatography
separation by biological binding interactions
wash
porous
bead elute
apply sample
protein of interest
Beads: Cellulose
agarose

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.
Tags

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.
His tags
28
Imidazole
N3H+-OOC
Histidine
• His and imidazole structure similarities
• Imidazole competes with His for Ni2+
sites

28.
GST Tag

29.
Strep Tag
PBS buffer (wash)Removal
indicated by
Red to yellow

30.
FLAG Tag

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.
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.
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.
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