11. Centrifuge for 5 min at 10,000 Â g to concentrate the pellet.
Aspirate the remaining liquid.
12. Resuspend the pellet in 20 ml of PBT buffer and transfer to a
50 ml conical tube and centrifuge for 5 min at 16,000 Â g to
pellet insoluble debris.
13. Transfer the supernatant to a new tube. Estimate the concentration by A 268 (OD 268 ¼ 1 corresponds to approximately
5 Â 10
12 phage/ml). The concentration of the library can be
better estimated by making a 10Â dilution series of the library,
infecting log phase OMNImax E. coli and plating on LB/carb
plates.
14. Add ultrapure glycerol to a final concentration of 10% and store
the library at À80
C (see Note 4). The quality and coverage of
the constructed library should be analyzed through NGS as
described in Subheading 3.8.
3.6 Bait Protein
Expression
and Purification
We typically use GST- or MBP-tagged PDZ domains, and remove
nonspecific binding phages and potential GST/MBP binders
through preselections against immobilized GST/MBP. For ProPPD, 10 μg protein is needed per selection day and at least half the
amount is needed for pooled phage ELISA. For 4 days of selection,
the minimum amount of protein needed is 60 μg. However, we
recommend performing triplicate selections, and to purify
0.5–1 mg protein of at least 90% purity. Below is a protocol for
batch purification of His-GST/MBP-tagged PDZ domains using
Ni-NTA beads starting from the expression construct in a pETM
vector.
3.6.1 Protein Expression
1. Transform the expression plasmid into E. coli BL21 (DE3).
2. Pick a single colony in to a 5 ml 2YT culture and grow it
overnight with shaking.
3. Transfer 1 ml of overnight culture into a 250 ml flask with
50 ml 2YT media supplemented with appropriate antibiotic.
4. Incubate at 37
C for approximately 2–3 h with shaking.
5. Measure OD 600 .
6. Induce protein expression at OD 600 ¼ 0.6-0.8 with 1 mM
IPTG final concentration.
7. Lower the temperature to 30
C and allow protein expression
to proceed for 4 hours.
8. Centrifuge cultures at 4
C for 20 min at 3000 Â g. Discard the
supernatant.
9. Freeze the bacterial pellet at À20
C.
Proteomic Peptide-Phage Display of PDZ Domains
51
Aspirate the remaining liquid.
12. Resuspend the pellet in 20 ml of PBT buffer and transfer to a
50 ml conical tube and centrifuge for 5 min at 16,000 Â g to
pellet insoluble debris.
13. Transfer the supernatant to a new tube. Estimate the concentration by A 268 (OD 268 ¼ 1 corresponds to approximately
5 Â 10
12 phage/ml). The concentration of the library can be
better estimated by making a 10Â dilution series of the library,
infecting log phase OMNImax E. coli and plating on LB/carb
plates.
14. Add ultrapure glycerol to a final concentration of 10% and store
the library at À80
C (see Note 4). The quality and coverage of
the constructed library should be analyzed through NGS as
described in Subheading 3.8.
3.6 Bait Protein
Expression
and Purification
We typically use GST- or MBP-tagged PDZ domains, and remove
nonspecific binding phages and potential GST/MBP binders
through preselections against immobilized GST/MBP. For ProPPD, 10 μg protein is needed per selection day and at least half the
amount is needed for pooled phage ELISA. For 4 days of selection,
the minimum amount of protein needed is 60 μg. However, we
recommend performing triplicate selections, and to purify
0.5–1 mg protein of at least 90% purity. Below is a protocol for
batch purification of His-GST/MBP-tagged PDZ domains using
Ni-NTA beads starting from the expression construct in a pETM
vector.
3.6.1 Protein Expression
1. Transform the expression plasmid into E. coli BL21 (DE3).
2. Pick a single colony in to a 5 ml 2YT culture and grow it
overnight with shaking.
3. Transfer 1 ml of overnight culture into a 250 ml flask with
50 ml 2YT media supplemented with appropriate antibiotic.
4. Incubate at 37
C for approximately 2–3 h with shaking.
5. Measure OD 600 .
6. Induce protein expression at OD 600 ¼ 0.6-0.8 with 1 mM
IPTG final concentration.
7. Lower the temperature to 30
C and allow protein expression
to proceed for 4 hours.
8. Centrifuge cultures at 4
C for 20 min at 3000 Â g. Discard the
supernatant.
9. Freeze the bacterial pellet at À20
C.
Proteomic Peptide-Phage Display of PDZ Domains
51