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 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.
 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?
Expression Choices
Features of expression vector

•Origin of replication
•Drug resistance
marker
•A promoter
•Transcription
terminator
•Restriction sites
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
pBAD
 Induction by addition of
arabinose
 Sugar binds to Ara protein
 Complex binds and initiates
RNA polymerase
 Good choice for toxic
proteins
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
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 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.
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
Pros and Cons of E.coli system

Advantages Disadvantages
High yield No post-translational
modification
easier scale up
Large proteins may be
inexpensive media difficult to express
Many expression Membrane proteins may not
plasmid fold well
Fast growth
 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.
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.
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
Yeast Expression strains
S. cerevisae 釀酒酵母

Pichia pastoris嗜甲醇酵母菌 Kluyveromyces lactis 乳酸克魯維酵母

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.
Pros and Cons of yeast system

Advantages Disadvantages
•Lacks detectable endotoxins.
•Gene expression less
•Fermentation relatively easily
inexpensive. controlled.
•Facilitates glycosylation •Glycosylation not
and formation of identical to mammalian
disulphide bonds. systems.
•Only 0.5% native proteins
are secreted so isolation of
secreted product is
simplified.
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
 Purification methods
SDS-PAGE
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
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
 Affinity chromatography
separation by biological binding interactions
protein of interest

apply sample

porous
bead wash elute

Beads: Cellulose
agarose
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.
Tags
His tags
His tags are typically a series of 6 histidines added to the C or N terminus of a
recombinant protein

• His tag and column interaction

Ni

Resin (IMAC nickel)

His-tagged
Recombinant
Protein
27
His tags
•His and imidazole structure similarities
•Imidazole competes with His for Ni2+ sites

Histidine Imidazole
-OOC N3H+

28
GST Tag
 Strep Tag

Removal PBS buffer (wash)

indicated by
Red to yellow
FLAG Tag
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.
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
 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