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

Patching

Grow in ampicillin agar for selection

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

Cloning Strategy:(Overview)

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:

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

Size: 20bp

OLIGO Start Length Sequence

Forward Primer

182 20bp 5’ ATG CCT TCT GAG ACC CCC CA 3’

Reverse Primer

1818 20bp 5’ TCA CTT CTG GTC CCG CTC CT 3’

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.

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 = 3Tm = (4+4)2 + (3+9)4 = 64ºCTherefore, the annealing temperature for forward primer is 64ºC - 2ºC = 62ºCReverse primer : 5’ TCA CTT CTG GTC CCG CTC CT 3’A = 1, T = 7, C = 9, G = 3Tm = (1+7)2 + (3+9)4 = 64ºCTherefore, the annealing temperature for reverse primer is 64ºC - 2ºC = 62ºCBoth primers have same melting & annealing temperature, therefore ideal annealing temperature for PCR = 62ºC

RT-PCR MethodologyConsists 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 ?

1. 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 endSolution : Add linkers

Characteristic of Taq Polymerase: One Adenine (A) will be added to each 3’ end of each strand of the PCR product.

A

A

5’

5’3’

3’

Definition: Linkers are short, artificially synthesized pieces of DNA containing a restriction enzyme site. EcoRI restriction sites

T4 DNA Polymerase

T4 DNA Polymerase

T

T

dTTPs

dTTPs

Linker ligation cont…Blunt ended PCR product

EcoRI RE siteEcoRI RE site

T4 DNA Ligase

Ligation

GENE OF INTERESTEcoRI RE site EcoRI RE site

Cut with EcoRI RE

GENE OF INTEREST

Sticky ends (EcoRI RE site)

Choosing vector:

• 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.

Criteria:

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 culture them for PCR screening.

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 PCR product.

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 NC

Position of wells

1.6 kb

MSample

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

Growth and culture of recombinant E.Coli

Diagram shows how the E.Coli is grown in the culture

Centrifuge the product at 10,000 rpm at temperature 4C.Remove supernatant and add lysate buffer

Analyzed the expression of gene by running SDS-PAGE

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

SDS-PAGE RESULT61kDa (POI)

PROTEIN PURIFICATION

use : His-Patch ThioFusion Expression System

Gene inserted into E.coli

Induce protein expression - IPTG3 hours

centrifuged

(10,000 rpm,4ºC)

Remove supernatant – add lysate buffer

Run SDS-PAGE -confirm fusion protein

RESULT :Extra protein band

PURIFY :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

HOW ?1. Protein binds to the nickel column

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

EnterokinaseAfter 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 InterestPurification 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 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.

Example:

pGEX

Glutathione-S-Transferase (GST) fusions

pGEX

tac

pGEX

tac

•IPTGinduction

•High level expression

pGEX

tac

•IPTGinduction

•High level expression

GST Foreign gene

GST comes fromSchistosoma mansoni

pGEX

tac

•IPTGinduction

•High level expression

GST Foreign gene

lacIq super repressor

pGEX

tac

•IPTGinduction

•High level expression

GST Foreign gene

lacIq super repressorampR

PURIFICATION OF GST FUSION PROTEINS

PURIFICATION

• EASY

• AFFINITY CHROMATOGRAPHY

PURIFICATIONDETAILS

• 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

GST

FOREIGN PEPTIDE

FUSION PROTEIN BOUND TO GLUTATHIONE SEPHAROSE

glutathione

GST

FOREIGN PEPTIDE

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

proteasedialyse

secondglutathionecolumn

pure foreignpeptide in flowthrough -GST sticks

+ GST

foreign peptide

pure fusion protein + glutathione

pure fusion

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

NO

N N N N

HistidineImidazole

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

trcpromoter

pYA292

foreign gene

asd

asd complements asd host & is thus stable

•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)

Eight fusion protein expression vectors

Lane 1: whole cell lysates of induced cellsLane 2: whole cell lysates of uninduced cellsLane 3: soluble proteins with induction

Fusion Proteins Solubility Test

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