Expression of Foreign Proteins in

E. Coli
-A Practical approach

Navin Khanna
Overview of a practical approach
• Gene isolation: RTPCR; Linker ligation
• Choosing vector: pThio-His
• Vector ligation & transformation
• Orientation of insert: PCR
• Protein Expression
• Purification by Ni-NTA affinity
• Enterokinase cleavege
• SDS PAGE Analysis
• GST Vector, pQE and other fusion vectors
• Vector for toxic proteins
• WHEN TO CHOOSE OTHER EXPRESSION SYSTEMS?
Cloning Strategy:
(Overview)
Analysis of sequence

Design primer

RT-PCR / PCR

Adding (RE) EcoRI linkers

Ligation with EcoRI cut pThioHis A plasmid

Transformation into E.coli

Grow in ampicillin agar for selection

Patching
Cloning Strategy:
(Overview)
PCR screening for recombinant DNA

Grow the successful clones then

followed by IPTG induction to yield protein of interest.

Protein purification using ProBond™ purification kit from Invitrogen.

Cleavage of HP-Thioredoxin by using

EnterokinaseMax from Invitrogen

SDS-PAGE to confirm purification of protein of interest

Purified protein
 Protein Estimation (Molecular Weight)

• Analysis of sequence using DNA Strider software

- length : 1656 base pairs
- 3 base pairs = 1 amino acid = 110 Daltons
- The molecular weight of protein of interest

= (1656/ 3) x 110 Daltons

= 60610 Daltons
= approximately 61 kDa
 Design of primer
Definition: A short nucleic acid sequence containing a free 3’ hydroxyl
group that forms base pairs with a complementary template
strand and functions as the starting point for addition of
nucleotides to copy the template strand.

Purpose: To perform RT-PCR amplification of gene of interest

Size: 20bp

OLIGO Start Length Sequence

Forward
182 20bp 5’ ATG CCT TCT GAG ACC CCC CA 3’
Primer
Reverse
1818 20bp 5’ TCA CTT CTG GTC CCG CTC CT 3’
Primer
 Calculating Melting & Annealing
Temperature
• Melting temperature Tm = (A+T)2 + (G+C)4
• Annealing temperature = Tm – 2 or 5ºC
Forward primer : 5’ ATG CCT TCT GAG ACC CCC CA 3’
A = 4, T = 4, C = 9, G = 3
Tm = (4+4)2 + (3+9)4
= 64ºC
Therefore, the annealing temperature for forward primer is 64ºC - 2ºC
= 62ºC
Reverse primer : 5’ TCA CTT CTG GTC CCG CTC CT 3’
A = 1, T = 7, C = 9, G = 3
Tm = (1+7)2 + (3+9)4
= 64ºC
Therefore, the annealing temperature for reverse primer is 64ºC - 2ºC
= 62ºC
Both primers have same melting & annealing temperature, therefore
ideal annealing temperature for PCR = 62ºC
 RT-PCR Methodology
Consists of : 1. Synthesis of cDNA from RNA by reverse
transcription (RT).
2. Amplification of a specific cDNA by
polymerase chain reaction (PCR).

How does it work ?

• RT buffer, dNTPs, 2 DNA primers, Taq polymerase, Reverse

Transcriptase, and mRNA are added into a micro centrifuge
tube.

2. Incubated at 37ºC
 RT-PCR Methodology Cont…

In the tube:

With the synthesis of cDNA,

PCR can be performed as
usual.
Polymerase Chain Reaction (PCR)
 Linker ligation
PCR product : Blunt end Definition: Linkers are short,
artificially synthesized
Solution : Add linkers pieces of DNA containing a
restriction enzyme site.
EcoRI restriction sites

Characteristic of Taq Polymerase: One Adenine (A) will be added to

each 3’ end of each strand of the
PCR product.
dTTPs
5’ 3’ T4 DNA
T A Polymerase

A T
T4 DNA 3’ 5’ dTTPs
Polymerase
 Linker ligation cont…
Blunt ended PCR product
EcoRI RE site EcoRI RE site

T4 DNA Ligase

Ligation

EcoRI RE site GENE OF INTEREST EcoRI RE site

Cut with EcoRI RE

GENE OF INTEREST

Sticky ends (EcoRI RE site)

 Choosing vector:
Criteria:
• Capable of cloning & expression.
• Ori site that can be recognized by E.coli (Host).
• Specific marker for identification. (Eg. AmpR)
• Compatible RE sites. (Eg. EcoRI, XhoI)
• Promoter that can be recognized by E.coli for gene
expression. (Eg. T7, Trc)
• Has lacIq ORF codes for repressor; IPTG induction.
• ORF for His-patch to produce fusion protein for easy
purification. (Eg. Thioredoxin)
• Enterokinase site (EK site) to cleave fusion protein.
Choosing vector: (Invitrogen)
 Vector Ligation
• pThioHisA is cut with EcoRI restriction enzyme
to create the linearized plasmid.
• Calf Intestine Alkaline Phosphatase (CIAP) is
added to prevent self-ligation and re-ligation of
the plasmids.
• PCR product has EcoRI sticky ends.
• Gene of Interest & pThioHisA are ligated
together in a microfuge tube with T4 DNA ligase.
• There are 2 possible insert orientation of the
gene. (Non-directional cloning)
• Can be verified by PCR screening method.
 Recombinant plasmid

(GOI)
 Transformation
• Transforming the recombinant plasmids (ligation mix) into
E.coli (TOP10) host. The ligation mix contains the gene of
interest ligated in pThioHIS A vector.

Calcium chloride method

• E.coli cells have to undergo some form of physical or

chemical treatment to be made competent.
• Will enable the bacteria to take up foreign plasmid DNA;
GOI DNA.
• Loosen up the cell wall so that circular DNA molecules
can be slipped through while maintaining the cells’
viability.
Transformation
 Growth in ampicillin agar
• Cells which “took up” the plasmids will be
able to grow on medium containing
ampicillin whereas those fail to “take in”
the plasmids will not be able to grow on the
ampicillin agar.

• Colonies of bacteria are all descended from

a single cell, this allows us to identify which
colonies have our gene inserted in the
correct orientation by using the PCR
screening method.

• Colonies that grow on the ampicillin agar

can be patched to a new agar plate to
 PCR screening for recombinant
DNA.
Principle

• To confirmed the presence of GOI insert in pThio

His A vector. The cloning method we have followed
in this example is a non-directional cloning
method.
• Hence there are possibilities of getting the GOI
insert in two orientations in the pThio His A vector.
By using one primer specific for vector and another
primer specific for the insert; it is possible to
identify the clone and its insert orientation.
• trc promoter present at pThio HIS A vector and the
reverse primer is present in POI gene.
• In the forward orientation, they will produce PCR
product.
• In reverse orientation, they will not produce our
POI gene in forward orientation POI gene in reverse orientation

trc primer Reverse primer trc primer Reverse primer

1656bp product NO product

 Result
• From the agarose gel electrophoresis, we observe that
there is no band seen in the negative control lane.
• The target PCR product was seen in the positive control
lane and sample lane. It’s indicating that our gene is
inserted in forward orientation. Hence, we will use this
gene to further our cloning stimulation project.

M PC Sample NC M
Position of wells

1.6 kb
Growth and culture of recombinant E.Coli

After PCR screening technique 

Confirmed E.coli hosts with correctly inserted POI gene are picked 
from the colonies patching dish.

Transferred into a flask containing LB broth and ampicillin
 for the culture to grow

Take 5 ml of the solution 

Transfer into a bigger flask containing 500ml of nutrient for 3 hours at 
37ºC

Add 0.4mM IPTG to induce protein expression
 
Diagram shows how the E.Coli is grown in the culture
Centrifuge the product at 10,000 rpm at temperature 4°C.

Remove supernatant and add lysate buffer

Analyzed the expression of gene by running SDS­PAGE 

Diagram shows how we lysed the bacteria
 SDS-PAGE
Objective : to confirm that we obtained the fusion protein before           
                            
                  purifying through the ProBond™ resin

Result : one additional band appear in lane S indicate the 
  
   existing of fusion protein 
C = control (lysed E.coli)
S = sample (lysed recombinant E.Coli)
C S

SDS­PAGE RESULT
61kDa
(POI)
 PROTEIN
PURIFICATION
• use : His-Patch ThioFusion Expression System

Gene inserted into E.coli

Induce protein expression - IPTG centrifuged

3 hours (10,000 rpm,4ºC)

Run SDS-PAGE Remove supernatant

-confirm fusion protein – add lysate buffer

RESULT : PURIFY :
Extra protein band ProBond™ resin (IMAC)
 FUSION PROTEIN

Protein of interest bound to resin

Resin were eluted with imidazole

Harvest fusion protein

 ENTEROKINASE

USE : To cut HP-Thioredoxin fusions

1. Protein binds to the nickel column

HOW ?
2. Elute the protein – imidazole
3. Treated with enterokinase – cleave
HP-Thioredoxin tag
4. Running the protein over the nickel
column again – freed the protein
5. Get the purify protein - POI
 Enterokinase
After the fusion protein is purified or partially purified by affinity
chromatography with ProBond resin, osmotic shock or other purification
method, it is then digested with enterokinase enzyme to release HP-thioredoxin
from fusion protein.
As a result, only our protein of interest will be extracted.
The enterokinase site in pThioHis vector permits the release HP-thioredoxin
from our protein.
EnterokinaseMax™, by Invitrogen is recombinant preparation
of the catalytic subunit of the enterokinase enzyme with a high
specific activity.
It recognizes the amino acid sequence Asp-Asp-Asp-Asp-Lys
and hydrolyzes the peptide bond between lysine and the next
amino acid.
 GET PROTEIN
OF INTEREST

Remove the HP-Thioredoxin fusion partner

from protein

EnterokinaseMax™
Cleave the fusion protein
= recombinant preparation of the catalytic
subunit of bovine enterokinase

Get only the protein of

interest
 SDS-PAGE
TO CONFIRM

WHY ? to make sure that the right protein is produced

HOW ? Protein is loaded into SDS-PAGE

Run the gel

Result can be seen using Coomassie

blue stain.

RESULT Produce one band – Approx. 61kDa (protein weight)

 Detecting Protein of Interest
Purification stages and chromatography profiles are
analyzed by SDS-PAGE.
This is important to assay overall purity and
enrichment the desired target protein.
The protein is Example:
loaded in the SDS-
PAGE and stained
using Coomassie
blue stain.
One band should
be appeared
This proved that
we had
successfully
cloned the gene
and expressed the
protein of interest
as well.
 pGEX

Glutathione-S-Transferase
(GST) fusions
 pGEX

tac
 pGEX

•IPTG
induction
tac
•High level
expression
 pGEX
GST comes from
Schistosoma
mansoni GST Foreign gene
•IPTG
induction
tac
•High level
expression
 pGEX

GST Foreign gene

•IPTG
induction
tac
•High level
expression

lacIq super repressor

 pGEX

GST Foreign gene

•IPTG
induction
tac
•High level
expression

lacIq super repressor

ampR
PURIFICATION OF GST
FUSION PROTEINS
 PURIFICATION
• EASY
• AFFINITY CHROMATOGRAPHY
 PURIFICATION
DETAILS
• GROW SAY 1L CULTURE TO MID LOG
PHASE
• ie OD260 = 0.4 – 0.7
• SPIN DOWN CELLS
• SONICATE IN PRESENCE OF
PROTEASE INHIBITORS
• POUR LYSATE OVER GLUTAHIONE
SEPHAROSE BEADS IN A COLUMN
GLUTATHIONE
SEPHAROSE

glutathione

SEPHAROSE
FUSION PROTEIN

FOREIGN PEPTIDE

GST
FUSION PROTEIN BOUND TO
GLUTATHIONE SEPHAROSE
FOREIGN PEPTIDE

GST

glutathione

SEPHAROSE
 PURIFICATION
• WASH COLUMN EXTENSIVELY
• ELUTE WITH REDUCED GLUTATHIONE
• RESULTS IN PURE GST FUSION
PROTEIN
COMPETITIVE ELUTION WITH
GLUTATHIONE

SEPHAROSE
 RESULT OF AFFINTY PURIFICATION AND
REMOVAL OF GST MOIETY

pure fusion protein

+ glutathione foreign peptide
+ GST
protease
dialyse
pure fusion second
glutathione
column

pure foreign
peptide in flow
through -
GST sticks
pQE VECTORS (Qia Express)
• Hex-histidine tag system
• Produce peptides with 6 histidines fused to
N or C terminus
• Allows Nickel Chelate Affinity
Chromatography
pQE VECTORS (Qia Express)
• Promoter
– engineered from phage T5 + lac operator
– 2 operator sites
– IPTG inducible
– Expression in host containing multiple copies
of pREP4 which has lacI
pQE VECTORS (Qia Express)
• Interaction between Ni2+ resin called
NTA is very strong and chemically resilient
– every Ni2+ binds 2 his residues in a non-
conformation dependent manner
– therefore resists strong denaturants eg 6M
guanidium HCl
 IMAC

IMAC :
Immobilized Metal Affinity Chromatography

• principle that is used in ProBond™ resin

• advantages :
• - only takes 2 – 3 hours
• - simple steps
• steps – same as above
• after purify – treat with special protease (enterokinase)
pQE VECTORS (Qia Express)
• Elution
– competitive with imidazole

O
N

N N N
N
Imidazole
Histidine
 pQE VECTORS (Qia Express)
• Removal of His tag?
– not necessary usually
– many hundreds of proteins purified with no
effect on structure
– not immunogenic
 EXPRESSION SYSTEMS
MOST USE PLASMIDS
– PROBLEMS
• INSTABILITY
• TOXICITY
• pET DUAL PLASMID
• BALANCED LETHAL
 BALANCED – LETHAL
SYSTEM
• OTHER SYSTEMS DESCRIBED CARRY
ANTIBIOTIC RESISTANCE-UNDESIREABLE
• THESE VECTORS COMPLEMENT LETHAL
DELETION IN HOST
• GENE FOR B-ASPARTATE SEMI-ALDEHYDE
DEHYDROGENASE OR asd
• asd MUTANTS HAVE ABSOLUTE
REQUIREMENT FOR DIAMINOPIMELIC ACID
(DAP) A CONSTITUENT OF THE CELL WALL
• THERE IS NO DAP IN MAMMALS
 Balanced Lethal

foreign gene
trc
promoter

pYA292
asd

asd complements asd ∆ host & is thus stable

Eight fusion protein expression vectors
•His (histidine)
•Trx (thioredoxin)
•NusA (NusA protein) : pET-43.1a (Novagen)
•CAP (cellulose-associated protein) : pET-35b2 (Novagen)
•CBP (calmodulin binding protein) : pET-22b+ (Novagen)
•Intein (chitin binding tag) : pTYB11(NEB)
•MBP (maltose-binding protein) : pMAL-C2XC (NEB)
•GST (glutathione S-transferase) : pGEX-4T (Pharmacia)
Fusion Proteins Solubility Test

Lane 1: whole cell lysates of induced cells

Lane 2: whole cell lysates of uninduced cells
Lane 3: soluble proteins with induction
 When not to use E.Coli?
• When you require
– Glycosylation
– Phosphorylation
– Cys rich proteins
– No N terminus Methionine
– Acetylation
– If the protein is too large or too small or too
hydrophobic or no way to re-nature IBs