Transcript Mudassar Presentation - Aller

Expression and Purification of
mutant of the Sensor domain
of EnvZ
Mudassar
Two Component System
The system used by bacteria to adapt to changes
in its environment. It is composed of two
components.
• Histidine Kinase:
Senses the environmental changes, such
as starvation of phosphate and nitrogen, adoption
to new carbon sources, limitations to oxygen and etc.
• Response Regulator:
Mediates a response by binding to specific
genes and altering their expression.
Two Component System
Two Component Regulatory System
signal causes sensor
protein to change
conf ormation
signal
sensor protein then
auto-phosphorylates
out side
inner
membrane
cyt oplasm
sensor
sensor
sensor
P
ATP
regulator
ADP
P
regulator
active f orm of regulator binds DNA
in regulatory region of target genes
regulator
P
structural genes
DNA
gene 1
promot er
gene 2
gene 3
EnvZ - OmpR
• EnvZ:
Acts as a sensor and senses osmolarity changes
in the environment.
• OmpR:
Reponse regulator, mediates a response by
binding to the promoter region of genes that code
for proteins that form pores in the outer membrane
of bacteria and let hydrophilic molecules in the cell.
Sub-Cloning
• PCR
• Restriction Digestion
• Ligation
PCR
• To amplify our gene of interest.
• To make L43A mutation.
• To introduce restriction site.
PCR
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PCR
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Denatured or melted
PCR
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Anealing primers
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PCR
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Extending Primers
PCR
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Denatured
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PCR
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Anealing primers
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PCR
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Extending primers
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PCR
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Vector
• To introduce a gene of interest into bacteria.
• Hallmarks:
- Multi cloning site.
- Selection marker.
- Promoter.
Restriction Digestion
Gene of interest
Bam H1
Nde 1
Nde 1
Bam H1
Vector
(pET 15b)
Selection Marker
Ligation
Nde 1
Vector
Bam H1
Insert (PCR product)
Vector
Ligation
Resulting vector
T7 Promoter
6 His tag
Nde 1
Selection Marker
Gene of Interest
Bam H1
Bacterial Growth
• Introduce the vector in bacteria.
• Grow bacteria.
Bacterial Growth
Transformation
Put the CaCl2
Bacterial cell
Bacterial DNA
Pores
Plasmid DNA
Bacterial Growth
Bacterial cell
Bacterial DNA
Bacterial Growth
Growing Culture
Spread transformed bacterial cells on the LB plate with selection
drug and grow overnight.
Bacterial Growth
Growing Culture
Liquid LB medium
with bacteria in it
Bacterial Growth
Growing Culture
Phase 3
Phase 2
OD600
IPTG induction
Phase 1
Time
Bacterial Growth
Pellet
Purification
•
•
•
•
Lysis.
Affinity Chromatography.
Cleavage of His tag.
Size Exclusion Chromatography.
Purification
Bacteria expressing sensor domain
Bacterial DNA
Plasmid DNA
Bacterial protein
Sensor domain
Lysis
Our protein is not soluble in bacterial cytoplasm.
Therefore the lysis would have two different parts:
- Native lysis.
- Denaturing lysis.
Lysis
Native Lysis:
- to isolate cell wall and inclusion bodies.
• Sonication.
• Centrifuge.
Lysis
Denatured Lysis:
- to denature and solublize our protein away
the debris material.
• Cell wall material is further broken up by liquid
nitrogen and hot water baths.
• Pellet is resuspended in the lysis buffer containing
6M guandine, and sonicated to further solublize
the protein.
• Spin down the denaturing lysis buffer, cell wall
and debris will pellet at the bottom and our protein
is in the soluble supernatant.
Affinity Chromatography
• To purify our protein from other bacterial proteins.
• The column contains a compound of Ni that helps
purify the protein.
• Histidine in the protein forms a physical bond with
Ni, thus binding the column.
Affinity Chromatography
6 Histidine residues in a row.
His His
His
His
His His
Ni
Agarose
Affinity Chromatography
Bind denatured protein to the Ni-Agarose
column on the shaker for at least one hour.
Wash it with the wash buffer to remove:
• anything that did not bind the column, and
• anything that bound very weakly.
Only our sensor domain with a 6X His tag
would have bound the column strongly.
Elute with the elution buffer with little amount
of imidazole in it, and collect fractions.
Affinity Chromatography
• Elute the column with elution buffer, increasing the
amount of imidazole in it each time.
• Imidazole replaces His on the column and
the protein is released.
• All the protein on the column would come out, and
be collected into fractions.
• Run samples from the fractions on the gel and pool
together the ones that have only our protein in it.
Affinity Chromatography
36.5 kDa
31 kDa
21.5 kDa
14.4 kDa
6 kDa
supernatant
pellet
Denaturing
lysis
Ni-NTA column
Wash
Elution fractions
Refolding
• Our protein was insoluble therefore we had to
denature our protein in order to purify it.
• To conduct an analysis on a protein it has to be in
its natural refolded state.
Refolding
To refold we put the protein in the refolding buffer
that does not contain any Guanidine and about 30%
of the protein refolds and the rest comes out of
solution.
Cleavage of His tag
His tag is not part of the protein. It needs to be
removed in order to perform structural and
biophysical studies on the protein.
- Thrombin is used to remove the His tag.
Cleavage of His tag
Thrombine:
It is a protease, an enzyme that cleaves of the protein
at a specific recognition sequence.
When Thrombine is added in the solution it recognizes
the cutting site, located just after the His tag and cleaves
the his tag off.
Cleavage of His tag
Now we have Thrombin mixed in with our protein.
• To remove Thrombin we add benzamidine sepharose.
• Benzamidine will bind to the protease (Thrombin).
• Because benzamidine is linked to a resin
(sepharose), the benzamidine- Thrombin complex
can be separated from our protein by spinning it
down.
Size Exclusion Chromatography:
Now we have cleaved of the His tag from our protein.
But not all the the protein would be free of His tag.
Not all of the protein would be correctly folded either
To make sure that we have a very pure protein we
run it on S75 gel filtration column also called “Size
Exclusion Chromatography”
Size Exclusion Chromatography:
Small protein
Beads
Big protein
Size Exclusion Chromatography:
• The flow rate can be set.
• The fractions are collected run on the gel and
the ones that contain pure protein fractions are
pooled together.
• Now we have refolded, pure protein and
can be used for analysis.
Marker
Size Exclusion Chromatography:
Eluted fractions
Conclusion
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We were able to sub-clone a mutant of
the sensor domain of EnvZ.
Expression of the mutant was
successful and yielded high quantities
of a pure protein.
This protein will be used for further
structural and biochemical analysis.
Acknowledgement
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Dr. Mitsu Ikura
Dr. Ahmad Khorchid
Ms. Karen Thompson (Co-op Teacher,
Monarch Park Collegiate)