Cloning, Expression and Purification of sigma 54

Vanishri.B.R.BTL(H)041B.Sc.(Agricultural Biotechnology)

Principal investigator: Dr.Deepak T NairNational Centre for Biological Sciences

Guide : Dr. Deepti Jain

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.

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.

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.

• 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

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.

Sigma 54 protein

Work 1. Cloning of sigma 54 2. Expression check3. Protein purification4. Site directed mutagenesis(Expression check, Protein purification)5. Binding assay

CLONING

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

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

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 1494bpSize of pDJN1 is 5.5kbSize of pGEX6P1 is 5kb

Ligation

Confirmation of clonespGEX-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

Expression check

Expression check gel picture

1 2 3 4 5 6 7 8 9 10

97.4

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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 18⁰C ⁰C for 18 hours at150 rpm

Protein purification

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)

Affinity chromatography1 2 3 4 5 6 7 8 9

97.4

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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

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

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43

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20.1

14.3

mol wt of protein (σ54) 56KDa Concentration of protein 38mg/ml (volume 240µl)

After gel filtration SD200

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

Expression check of Sigma 54 (NTD)

1 2 3 4 5 6 7 8 9

97.4

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43

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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 18⁰c ⁰c for 18 hours at150 rpm

Protein loaded is 100ug in each well

C41DE3 cells used

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

Gel filtration

1 2 3 4 5 6 7 8 9 10

1 A7 57.7-53.7ml

2 A8 53.7-55.68

3 A9 55.68-57.66

4 A10 57.66-59.72

5 A11 59.72-61.69

6 MARKER

7 A12 61.69-63.67

8 B12 63.67-65.72

9 B11 65.72-67.70

10 B10 67.70-69.68

1 B5 77.66-79.72 ml

2 B4 79.72-81.69

3 B2 83.67-85.72

4 MARKER

5 B1 85.72-87.70

6 C1 87.70-89.68

7 C2 89.68-91.73

8 C4 93.71-95.69

9 B8 71.73-73.72

10 B7 73.72-75.69

1 2 3 4 5 6 7 8 9 10

29

20.1

14.3

29

20.1

14.3

mol wt of sigma 54 NTD protein 6.6kDa

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

BINDING ASSAY

8% Native PAGE

1 Sigma 54(15µg)

2 FleQ (15µg)

3 Sigma 54+FleQ(1:1 ratio)+AMPNPP

8% native PAGE1x TBE running buffer pH 8voltage :20V9 hour gel running in cold roomover night staining.

0.8% Agarose gel

1 2 3 4 5 1 Sigma 54(15µg)

2 FleQ(15µg)

3 Sigma 54+FleQ(1:1 ratio)

4 Sigma 54+FleQ+AMPNPP

5 Sigma 54+FleQ+ADP

Running Buffer: 1x TG+8mM MgCl2, pH7Voltage: 50VGel run time: 5 hourStaining: 1 hour

0.7% Agarose gel 1 2 3 4 5

1 Sigma 54(15µg)

2 FleQ(15µg)

3 Sigma 54+FleQ(1:1 ratio)

4 Sigma 54+FleQ+AMPNPP

5 Sigma 54+FleQ+ADP

Running Buffer: 1x TG+8mM MgCl2,pH7Voltage: 50VGel run time: 7 hourStaining: 45 minutes.

0.8% Agarose gel

1 2 3 4 5

1 Sigma 54(15µg)

2 FleQ(15µg)

3 Sigma 54+FleQ(1:1 ratio)

4 Sigma 54+FleQ+AMPNPP

5 Sigma 54+FleQ+ADP

Running Buffer: 1x TG+8mM MgCl2,pH7Voltage: 50VGel run time: 6 hourStaining: 45 minutes.

0.8% Agarose gel1 Sigma 54(15ug)

2 FleQ(15ug)

3 Sigma 54+FleQ(1:1 ratio)

4 Sigma 54+FleQ+AMPNPP

5 Sigma 54+FleQ+ADP

6 Sigma 54+FleQ+ATP1 2 3 4 5 6

Running Buffer: 1x TG+8mM MgCl2,pH7Voltage: 50vGel run time: 5 hourStaining: 45 minutes.

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.

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