3. Ligate the PCR fragment into pTWIN1 vector with T4 DNA
ligase according to the manufacturer’s instructions. This forms
the plasmid encoding the PDZ domain fragment fused to the
intein, pTWIN1-His 7 (Δ N PDZ).
4. Transform 1 μL of ligation mixture into 10 μL of chemically
competent E. coli cloning strain, TOP10. After growing the
culture in S.O.C. media for 45 min, apply 50 μL of cells onto an
LB agar plate with 100 μg/mL ampicillin. Allow colonies to
form overnight in an incubator at 37
C.
5. Prepare 5 mL of overnight culture in LB medium with
100 μg/mL ampicillin by inoculating one colony from the
LB-ampicillin plate. Allow the culture to grow overnight in a
shaking incubator at 37
C at 200 rpm.
6. Isolate plasmid, pTWIN1-His 7 (Δ N PDZ), from overnight culture with a DNA miniprep kit, according to manufacturer’s
instructions. Verify plasmid with DNA sequencing and store at
À20
C.
3.2 Protein
Expression
The following steps can be applied to the expression of both the
N- and C-terminal recombinant fragments.
1. Transform 1 μL DNA (obtained in Subheading 3.1) into 10 μL
of chemically competent E. coli expression strain, BL21 dE3
pLys. After growing in 100 μL of S.O.C. media for 45 min at
37
C and shaking at 200 rpm, spread 10 μL of cells onto an LB
agar plate containing 100 μg/mL ampicillin. Allow colonies to
form overnight in an incubator at 37
C.
2. Prepare the starting culture by inoculating one colony from the
LB-ampicillin plate into a 300-mL flask containing 100 mL of
sterile LB broth and allow the culture to grow overnight in a
shaking incubator at 37
C and shaking at 200 rpm.
3. Measure the optical density of the starting culture at the wavelength of 600 nm (OD 600 ) with a UV spectrophotometer. Add
starting culture to prewarmed 5 L flasks containing 1 L LB
broth with 100 μg/mL ampicillin to target a starting OD 600
value of 0.1.
4. Allow culture to grow in a shaking incubator at 37
C and
200 rpm until the OD 600 value reaches between 0.4 and 0.8.
Induce expression by adding IPTG stock to target a final
concentration of 1 mM to the culture. Then allow culture to
grow for another 4 h at 37
C and shaking at 200 rpm (see Note
4).
5. Harvest cells by centrifuging the culture at 10,000 Â g for
10 min at 4
C. Discard supernatant and collect pellet. Store
cell pellets at À20
C for later processing or proceed directly
into the next protein purification steps.
Synthetic PDZ Domains
203
ligase according to the manufacturer’s instructions. This forms
the plasmid encoding the PDZ domain fragment fused to the
intein, pTWIN1-His 7 (Δ N PDZ).
4. Transform 1 μL of ligation mixture into 10 μL of chemically
competent E. coli cloning strain, TOP10. After growing the
culture in S.O.C. media for 45 min, apply 50 μL of cells onto an
LB agar plate with 100 μg/mL ampicillin. Allow colonies to
form overnight in an incubator at 37
C.
5. Prepare 5 mL of overnight culture in LB medium with
100 μg/mL ampicillin by inoculating one colony from the
LB-ampicillin plate. Allow the culture to grow overnight in a
shaking incubator at 37
C at 200 rpm.
6. Isolate plasmid, pTWIN1-His 7 (Δ N PDZ), from overnight culture with a DNA miniprep kit, according to manufacturer’s
instructions. Verify plasmid with DNA sequencing and store at
À20
C.
3.2 Protein
Expression
The following steps can be applied to the expression of both the
N- and C-terminal recombinant fragments.
1. Transform 1 μL DNA (obtained in Subheading 3.1) into 10 μL
of chemically competent E. coli expression strain, BL21 dE3
pLys. After growing in 100 μL of S.O.C. media for 45 min at
37
C and shaking at 200 rpm, spread 10 μL of cells onto an LB
agar plate containing 100 μg/mL ampicillin. Allow colonies to
form overnight in an incubator at 37
C.
2. Prepare the starting culture by inoculating one colony from the
LB-ampicillin plate into a 300-mL flask containing 100 mL of
sterile LB broth and allow the culture to grow overnight in a
shaking incubator at 37
C and shaking at 200 rpm.
3. Measure the optical density of the starting culture at the wavelength of 600 nm (OD 600 ) with a UV spectrophotometer. Add
starting culture to prewarmed 5 L flasks containing 1 L LB
broth with 100 μg/mL ampicillin to target a starting OD 600
value of 0.1.
4. Allow culture to grow in a shaking incubator at 37
C and
200 rpm until the OD 600 value reaches between 0.4 and 0.8.
Induce expression by adding IPTG stock to target a final
concentration of 1 mM to the culture. Then allow culture to
grow for another 4 h at 37
C and shaking at 200 rpm (see Note
4).
5. Harvest cells by centrifuging the culture at 10,000 Â g for
10 min at 4
C. Discard supernatant and collect pellet. Store
cell pellets at À20
C for later processing or proceed directly
into the next protein purification steps.
Synthetic PDZ Domains
203