3.7 ProP-PD
Selection Against PDZ
Domains
The P8 protein used for C-terminal peptide phage display is permutated as compared to the wild-type p8 protein [12]. This makes
the display system less suited for high-throughput selections
described elsewhere [14], and we therefore follow a lower throughput protocol for C-terminal ProP-PD against PDZ domains. There
is typically a tenfold enrichment of binders per day of selection, and
we recommend to perform at least four consecutive days of selections, and to evaluate the success of the selection through pooled
phage ELISA the fifth day, although it is possible to delay the
pooled phage ELISA to the following week.
3.7.1 Day 0
1. Coat a 96-well MaxiSorp plate with purified bait protein (one
well per replicate selection. To each well, add 10 μg GST/
MBP-tagged PDZ domain in 100 μl PBS. In parallel, coat a
negative control plate with 10 μg GST/MBP. One well of
immobilized bait protein per replicate is typically enough for
selection against ProP-PD libraries (diversity in the order of
10,000 to 1 Â 10
6 ). As a rule of thumb, the number of phages
used should surpass 1000 times the library diversity, and the
phage concentration should not be more than 10
13 phage/ml.
2. Cover the plates with plastic seals and incubate overnight at
4
C with shaking. Note that immobilization also can be performed for 2 h at room temperature if needed.
3. Start an overnight culture of E. coli OmniMAX: Inoculate a
single colony E. coli OmniMAX in 10 ml 2YT supplemented
with 10 μl 10 mg/ml tetracycline. Incubate overnight at 37
C,
200 rpm. Store the stock culture at 4
C for the duration of the
phage selection protocol.
3.7.2 Selection Day 1
1. Inoculate 3 Â 10 ml 2YT in 50 ml Falcon tubes with 20 μl of
the stock E. coli OmniMAX culture. Culture 1 should be
supplemented with 10 μl tetracycline, and will be used for
elution of bound phages, Culture 2 and culture 3 should be
supplemented with 10 μl carbenicillin or 10 μl kanamycin, for
control of preinfection of library phage or M13KO7 helper
phage, respectively. Incubate the cultures at 37
C with
shaking.
2. Remove the protein solutions from the MaxiSorp plates and
block each well with 200 μl blocking solution for 1 h at 4
C.
3. Prepare naı ¨ve phage library from glycerol stocks: Calculate the
amount of library needed for the selection (1000 Â library
diversity  number of wells). Withdraw the required amount
of phage library from the stock solution, and dilute it 10 times
in PBS. Precipitate the library by the addition of 1/5 final
volume of PEG/NaCl. Incubate for 10 min on ice. Centrifuge
for 10 min at 17,000 Â g. Remove the supernatant by pouring.
Proteomic Peptide-Phage Display of PDZ Domains
53
Selection Against PDZ
Domains
The P8 protein used for C-terminal peptide phage display is permutated as compared to the wild-type p8 protein [12]. This makes
the display system less suited for high-throughput selections
described elsewhere [14], and we therefore follow a lower throughput protocol for C-terminal ProP-PD against PDZ domains. There
is typically a tenfold enrichment of binders per day of selection, and
we recommend to perform at least four consecutive days of selections, and to evaluate the success of the selection through pooled
phage ELISA the fifth day, although it is possible to delay the
pooled phage ELISA to the following week.
3.7.1 Day 0
1. Coat a 96-well MaxiSorp plate with purified bait protein (one
well per replicate selection. To each well, add 10 μg GST/
MBP-tagged PDZ domain in 100 μl PBS. In parallel, coat a
negative control plate with 10 μg GST/MBP. One well of
immobilized bait protein per replicate is typically enough for
selection against ProP-PD libraries (diversity in the order of
10,000 to 1 Â 10
6 ). As a rule of thumb, the number of phages
used should surpass 1000 times the library diversity, and the
phage concentration should not be more than 10
13 phage/ml.
2. Cover the plates with plastic seals and incubate overnight at
4
C with shaking. Note that immobilization also can be performed for 2 h at room temperature if needed.
3. Start an overnight culture of E. coli OmniMAX: Inoculate a
single colony E. coli OmniMAX in 10 ml 2YT supplemented
with 10 μl 10 mg/ml tetracycline. Incubate overnight at 37
C,
200 rpm. Store the stock culture at 4
C for the duration of the
phage selection protocol.
3.7.2 Selection Day 1
1. Inoculate 3 Â 10 ml 2YT in 50 ml Falcon tubes with 20 μl of
the stock E. coli OmniMAX culture. Culture 1 should be
supplemented with 10 μl tetracycline, and will be used for
elution of bound phages, Culture 2 and culture 3 should be
supplemented with 10 μl carbenicillin or 10 μl kanamycin, for
control of preinfection of library phage or M13KO7 helper
phage, respectively. Incubate the cultures at 37
C with
shaking.
2. Remove the protein solutions from the MaxiSorp plates and
block each well with 200 μl blocking solution for 1 h at 4
C.
3. Prepare naı ¨ve phage library from glycerol stocks: Calculate the
amount of library needed for the selection (1000 Â library
diversity  number of wells). Withdraw the required amount
of phage library from the stock solution, and dilute it 10 times
in PBS. Precipitate the library by the addition of 1/5 final
volume of PEG/NaCl. Incubate for 10 min on ice. Centrifuge
for 10 min at 17,000 Â g. Remove the supernatant by pouring.
Proteomic Peptide-Phage Display of PDZ Domains
53