Phd Course Work Drkhanna

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 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’

• •

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 Solution : Add linkers EcoRI restriction sites

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

Characteristic of Taq Polymerase: One Adenine (A) will be added to each 3’ end of each strand of the PCR product. 5’ 3’ T4 DNA dTTPs

T4 DNA Polymerase

T

A

A

T

3’

Polymerase

5’

dTTPs

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 trc primer

Reverse primer

1656bp product

POI gene in reverse orientation trc primer

Reverse primer

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 Position of wells 1.6 kb

PC

Sample NC

M

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) S C

SDS­PAGE RESULT

61kDa (POI)

PROTEIN PURIFICATION • use : His-Patch ThioFusion Expression System Gene inserted into E.coli centrifuged

Induce protein expression - IPTG 3 hours

(10,000 rpm,4ºC)

Run SDS-PAGE -confirm fusion protein

Remove supernatant – add lysate buffer

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

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 ?

HOW ?

to make sure that the right protein is produced

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 loaded in the SDSPAGE 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

•IPTG induction

tac •High level expression

pGEX GST comes from Schistosoma mansoni GST

•IPTG induction

tac •High level expression

Foreign gene

pGEX

•IPTG induction

GST

Foreign gene

tac •High level expression

lacIq super repressor

pGEX

•IPTG induction

GST

Foreign gene

tac •High level expression

ampR

lacIq super repressor

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

dialyse

foreign peptide protease pure fusion

+

GST

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 nonconformation 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

Histidine

N

N

Imidazole

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

trc promoter

foreign gene

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