phd course work drkhanna
DESCRIPTION
Presentation of Dr. Navin Khanna in Ph.D course work at ICGEB in 2004, Nov 9TRANSCRIPT
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Expression of Foreign Proteins in E. Coli
-A Practical approach
Navin Khanna
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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?
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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
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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)
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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
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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.
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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
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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
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RT-PCR Methodology Cont…
In the tube:
With the synthesis of cDNA, PCR can be performed as
usual.
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Polymerase Chain Reaction (PCR)
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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
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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)
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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:
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Choosing vector: (Invitrogen)
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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.
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Recombinant plasmid
(GOI)
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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.
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Transformation
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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.
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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.
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POI gene in forward orientation POI gene in reverse orientation
trc primer Reverse primer trc primer Reverse primer
1656bp product NO product
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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
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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
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Diagram shows how the E.Coli is grown in the culture
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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
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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)
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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)
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FUSION PROTEIN
Protein of interest bound to resin
Resin were eluted with imidazole
Harvest fusion protein
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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
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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.
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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.
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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
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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)
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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.
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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:
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pGEX
Glutathione-S-Transferase (GST) fusions
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pGEX
tac
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pGEX
tac
•IPTGinduction
•High level expression
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pGEX
tac
•IPTGinduction
•High level expression
GST Foreign gene
GST comes fromSchistosoma mansoni
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pGEX
tac
•IPTGinduction
•High level expression
GST Foreign gene
lacIq super repressor
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pGEX
tac
•IPTGinduction
•High level expression
GST Foreign gene
lacIq super repressorampR
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PURIFICATION OF GST FUSION PROTEINS
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PURIFICATION
• EASY
• AFFINITY CHROMATOGRAPHY
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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
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GLUTATHIONE SEPHAROSE
glutathione
SEPHAROSE
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FUSION PROTEIN
GST
FOREIGN PEPTIDE
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FUSION PROTEIN BOUND TO GLUTATHIONE SEPHAROSE
glutathione
GST
FOREIGN PEPTIDE
SEPHAROSE
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PURIFICATION
• WASH COLUMN EXTENSIVELY
• ELUTE WITH REDUCED GLUTATHIONE
• RESULTS IN PURE GST FUSION PROTEIN
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COMPETITIVE ELUTION WITH GLUTATHIONE
SEPHAROSE
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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
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pQE VECTORS (Qia Express)• Hex-histidine tag system
• Produce peptides with 6 histidines fused to N or C terminus
• Allows Nickel Chelate Affinity Chromatography
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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
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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
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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)
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pQE VECTORS (Qia Express)• Elution
– competitive with imidazole
NO
N N N N
HistidineImidazole
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pQE VECTORS (Qia Express)
• Removal of His tag?– not necessary usually– many hundreds of proteins purified with no
effect on structure– not immunogenic
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EXPRESSION SYSTEMS
MOST USE PLASMIDS– PROBLEMS
• INSTABILITY• TOXICITY
• pET DUAL PLASMID
• BALANCED LETHAL
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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
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Balanced Lethal
trcpromoter
pYA292
foreign gene
asd
asd complements asd host & is thus stable
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•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
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Lane 1: whole cell lysates of induced cellsLane 2: whole cell lysates of uninduced cellsLane 3: soluble proteins with induction
Fusion Proteins Solubility Test
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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