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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
Patching
Cloning Strategy:
(Overview)
PCR screening for recombinant DNA
Purified protein
Protein Estimation (Molecular Weight)
2. Incubated at 37ºC
RT-PCR Methodology Cont…
In the tube:
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
GENE OF INTEREST
(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.
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
RESULT : PURIFY :
Extra protein band ProBond™ resin (IMAC)
FUSION PROTEIN
EnterokinaseMax™
Cleave the fusion protein
= recombinant preparation of the catalytic
subunit of bovine enterokinase
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
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 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
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