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

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

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

Kluyveromyces lactis

Pichia pastoris

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

•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.

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

SDS-PAGE

purification methods

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

wash

porous

bead elute

apply sample

protein of interest

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

27

Resin (IMAC nickel)

Ni

His-tagged

Recombinant

Protein

• His tag and column interaction

His tags

28

Imidazole

N3H+-OOC

Histidine

• His and imidazole structure similarities

• Imidazole competes with His for Ni2+ sites

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

beads

Wash and elute

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