1. Cloning, Expression and
Purification of sigma 54
Vanishri.B.R.
BTL(H)041
B.Sc.(Agricultural Biotechnology)
Principal investigator: Dr.Deepak T Nair
National Centre for Biological Sciences
Guide : Dr. Deepti Jain
2. Pseudomonas aeruginosa
• As an opportunistic pathogen, P. aeruginosa has a remarkable
capacity to infect a large array of life-forms including mammals,
insects and plants .
• P. aeruginosa is known for its incredible metabolic diversity and
capacity for adaptation.
• It is known to form biofilms
• Lung Infections by this bacterium is what makes the situation worse
for patients of cystic fibrosis.
3. Transcription factors
• Transcription factors are protein that binds to specific DNA
sequences, thereby controlling the flow (or transcription) of genetic
information from DNA to messenger RNA.
• Transcription factors perform this function alone or with other
proteins in a complex, by promoting (as an activator), or blocking (as
a repressor) the recruitment of RNA polymerase to specific genes.
4. σ factor
• A sigma factor (σ factor) is a protein needed only for initiation of RNA
synthesis.
• It is a bacterial transcription initiation factor that enables specific
binding of RNA polymerase to gene promoters.
• RNA polymerase holoenzyme complex (consists of core RNA
polymerase and a sigma factor) executes transcription of a DNA
template strand.
5. • Transcription from σ54
has distinctive characteristics such as the
obligatory presence of a ATP-dependent transcriptional activator
which interacts with the sigma-54 subunit and a specific DNA
sequence.
• Hence, transcription from sigma-54 promoters can be entirely
turned-off in the absence of the transcriptional activator.
• σ54
is required for many functions such as nitrogen assimilation,
motility, transport of nutrients, formation of pili, mucoidy and cell-to-
cell signaling, phage-shock response and zinc tolerance.
σ54 factor
6. σ54 factor
• To proceed with initiation of transcription, the RNAP-sigma54
complex must participate in an interaction with a transcriptional
activator, involving nucleotide hydrolysis.
• The Pseudomonas databases predicted 22 proteins having the sigma-
54-ATP binding region.
• Among them, FleQ and FleR are involved in the biosynthesis of
flagellin, the major component of the single flagella of P. aeruginosa ,
PilR for pilin biosynthesis and AlgB that regulates alginate
exopolysaccharide production.
8. Work
1. Cloning of sigma 54
2. Expression check
3. Protein purification
4. Site directed mutagenesis(Expression check, Protein purification)
5. Binding assay
10. Cloning of sigma 54
• Plasmid and insert multiplication
• Digestion of plasmid and insert by restriction enzymes
• Ligation of insert into plasmid
• Transformation
• Growing on selective agar media
• Plasmid extraction from clone
• Confirmation
11. PCR amplification of sigma 54
1 1 kb ladder(1µl)-
2 PCR product(3µl)
3 PCR product(3µl)
4 PCR product(3µl)
1 2 3 4
Size of sigma 54 is 1494bp
12. Restriction Digestion of plasmids and
insert
1 2 3 4
1 1kb ladder(1µl)
2 Sigma 54(3µl) 32.307ng/µl
3 pDJN1(3µl) 37.50ng/µl
4 pGEX6P1(5µl) 14.5ng/µl
Size of sigma 54 is 1494bp
Size of pDJN1 is 5.5kb
Size of pGEX6P1 is 5kb
14. Confirmation of clones
pGEX-6-P1 σ54 clone (6.5 Kbp)pDJN1 σ54 clone (7Kbp)
1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8
Enzyme used for test digestion is Xho1 and incubation time is 2 hour at 37 ⁰C
16. Expression check gel picture
1 2 3 4 5 6 7 8 9 10
97.4
66
43
29
20.1
Expected mol wt of protein 82kDa (56+26)
C41DE3 pGEX-Sigma 54
Protein loaded is 100ug in each well
We got optimum expression in 0.2mM IPTG concentration at 18⁰C⁰C for 18 hours at150 rpm
18. Protein purification
• GST Affinity chromatography
• Prescission protease was added to the column to cleave the GST tag
and the free protein was eluted and concentrated and subjected to
size exclusion chromatography (SD200)
19. Affinity chromatography
1 2 3 4 5 6 7 8 9
97.4
66
43
29
20.1
1 Lysate sample
2 Flow through
3 Low salt buffer wash 1
4 high salt buffer wash
5 Low salt buffer wash 2
6 Marker
7 Protein
8 Glutathione wash
9 Filtrate
mol wt of sigma 54 protein 56kDa
20. 1 13 68-70
2 14 70-72
3 15 72-74
4 Marker
5 16 74-76
6 17 76-78
7 18 78-80
8 19 80-82
9 Lysate sample
1 2 3 4 5 6 7 8 9
97.4
66
43
29
20.1
14.3
mol wt of protein (σ54) 56KDa Concentration of protein 38mg/ml (volume 240µl)
After gel filtration SD200
21. Site directed mutagenesis of sigma54
• We added stop codon after 60 aminoacids. so, we made mutation in
such a way to express only N terminal region of sigma 54
• Confirmed the mutation by sequencing
• Then checked the expression and purified the sigma54 NTD
22. Expression check of Sigma 54
(NTD)
1 2 3 4 5 6 7 8 9
97.4
66
43
29
20.1
14.3
mol wt of sigma 54 NTD is 32.6kDa (6.6+26)
We got optimum expression in 0.5mM IPTG concentration at 18⁰c⁰c for 18 hours at150 rpm
Protein loaded is 100ug in each well
C41DE3 cells used
23. After GST affinity chromatography
1 Lysate sample
2 Flow through
3 Low salt buffer wash 1
4 high salt buffer wash
5 Low salt buffer wash 2
6 Marker
7 Protein
8 Glutathione wash
9 Filtrate
mol wt of sigma 54 NTD protein 6.6 kDa
97.4
66
43
29
20.1
14.3
1 2 3 4 5 6 7 8 9
26. 1 2 3
1 Glutathione wash
2 Protein
3 Marker
29
20.1
14.3
20.1
14.3
1 Protein
2 Marker
After concentration
Concentration of sigma54 NTD protein 6.26mg/ml,
total volume 300µl.
1 2
After passing through GST Column
33. Conclusion
• I cloned the gene for sigma 54 in pGEX6P1 and pDJN1. The condition
of optimum expression was determined and purified the protein
using affinity and size exclusion chromatography.
• I mutated the sigma 54- pGEX6P1 plasmid and determined the
optimum expression and I purified the protein.
• I carried out the binding studies using already purified fleQ and
sigma54. From the result it was not very clear about the interaction
between these two proteins.
34. Acknowledgement
• Dr.Veena S Anil and Dr.Krishnaprasad.B.T
• Dr. Deepak T Nair and Dr. Deepti Jain
• Jithesh K and Mary
• Neethu and Deepankar
• Lab 4 members
Editor's Notes
The sigma54 is having three regions with the N-terminal ~60 residues termed Region I, followed by a linker domain termed Region II, and a large C-terminal region with multiple individually folding functional domains termed Region III. The N-terminal of sigma54 interacts with the central ATPase domain of a variety of transcriptional activators. CBD interacts with the core RNA polymerase -12 and -24 DBD binds with corresponding up stream elements.
For cloning we should have sufficient amount of insert and plasmid. so,we multiplied our plasmids pgex and pdjn1 in DH5α cells and we amplified sigma54 insert by PCR
Here we amplified the sigma 54 gene from the genomic DNA by PCR and confirmed the amplification by running in the agarose gel. We can clearly see the amplification the gene in the 1.5kbp region. Further we carried out the purification of the pcr products by kit
We did sequential digestion of insert and plasmids using BamH1 and Xho1. From the gel it is clear that there is complete digestion of the plasmids and insert. After digestion, we purified both the plasmids and gene by gel extraction method then we went ahead with ligation
We did ligation of insert and plasmid then we transformed ligated plasmids into DH5 alpha cells. then again extracted the plasmids using mini prep kit.
we confirmed the presence of clones by digestion and running on the gel and further confirmed by sequencing. Then we went ahead with expression check. For expression check we used the pgex clone and the cell used was c41de3.
We got very good expression in 0.2mM IPTG concentration at 18⁰c condition. so,we used that condition for 5l culture.
For the purification we used GST affinity chromatography and followed by Size exclusion chromatography. We did on column cleavage for the removal of the GST tag and the free protein was collected and concentrated and subjected to Size exclusion chromatography in SD200.
Here is the gel after affinity chromatography and the 7 the lane showing the eluted protein. We concentrated and loaded onto SD200.
This is the gel filteration profile for the sigma 54 and the peak fractions were run it on sds page and gel shows the protein is pure and we concentrated lane 2 to 7 and flash freeze and stored. The purified protein was used for binding assay.
In order to check the interaction of N terminal domain of the sigma 54 with fleq, we have inserted a stop codon after 60 amino acids. The mutation was confirmed through sequencing and the we did expression check .
We have done the expression check for the sigma NTD in C41 cells and we got optimum expression in 0.5mM IPTG concentration at 18⁰c for 18 hours at150 rpm
Here is the gel after affinity chromatography and the 7th lane showing a faint band below the 14kda which is corresponding to the expected MW. We concentrated and loaded onto SD75. Since the sigma NTD is not having any aromatic residues, there will not be any absorption at 280nm.
This is the gel filtration profile for the sigma 54 NTD . The peak obtained may not be because the NTD, so we ran all the fractions in tricine gel, since it is a very low molecular weight protein.
This is the tricine gels for all those fractions and it is very clear that there is contamination of GST tag eventhough we attached GST column after the SD75. In order to remove the GST tag we again passed the solution through GST column and collected the flow through since the free protein wont bind to the column.
We have concentrated the protein and stored.
Through binding assay, I am checking the interaction of full length sigma with FleQ protein. For the binding assay we are using either native PAGE or agarose gel electrophoresis
Here we incubated the sigma 54 with Fleq in equimolar ratio in presense of AMPNPP and ran it in Native page. From the gel it is not clear that whether it is having binding or not. So we planned to repeat the experiment in agarose gels in the presence of ADP and AMPNPP
This is the gel for that experiment. In the fifth line, it seems to have some binding but it is not very clear. So we planned to repeat the same assay but running in a low percentage gel and running it for little longer time
Here also binding is not clear, so we planned to do the assay and running the gel by loading the sample in the middle of the gel.
Here the lines 3 4 5 showing thicker bands, but towards the anode region.
