3 Methods
3.1 Affimer Selection
by Phage Display
Target proteins can be biotinylated using chemical conjugation
methods, such as EZ-Link NHS-Biotin (ThermoFisher Scientific,
catalog no. 20217), following the manufacturer’s instructions.
Chemical biotinylation may block the binding sites on target proteins. Alternatively, target protein sequence can be tagged with a
biotin acceptor peptide (BAP) sequence (GLNDIFEAQKIEWHE), which is enzymatically biotinylated when expressed in
E. coli in the presence of biotin ligase.
3.1.1 Panning Round 1
Day 1
1. Block the streptavidin-coated wells with 300 μl per well of
2Â Blocking Buffer. Prepare four wells per selection—three
wells for pre-panning the phage and one well to immobilize
the target. Cover the wells with an adhesive plate seal and
incubate overnight at 37
C.
2. Set up an overnight culture of ER2738 E. coli cells. Pick a single
colony into 5 ml of 2YT medium with 12 μg/ml tetracycline
and incubate overnight in an orbital incubator at 37
C,
230 rpm.
Day 2
3. Wash the blocked wells 3Â with 300 μl of PBST and replace
with 100 μl per well of 2Â Blocking Buffer.
4. Immobilization of biotinylated target and deselection target
(if using) (see Note 1). Add 1 μg of biotinylated target to the
selection well (see Note 2). If required, add 1 μg of biotinylated
deselection target into pre-pan wells 2 and 3. Proceed immediately to step 5.
5. Pre-pan 1. To the first pre-pan well, add 5 μl of Affimer phage
library (~10
12 colony-forming units). Cover with a plate seal
and incubate for 40 min at room temperature on a vibrating
platform shaker.
6. Pre-pan 2. Remove the blocking buffer/deselection target
from pre-pan well 2. If deselection target was used, wash the
well 6Â with 300 μl of PBST. Transfer the phage from pre-pan
well 1 to pre-pan well 2. Cover with a plate seal and incubate
for 40 min at room temperature on the vibrating platform
shaker.
7. Pre-pan 3. Remove the blocking buffer/deselection target
from pre-pan well 3. If deselection target was used, wash the
well 6Â with 300 μl of PBST. Transfer the phage from pre-pan
well 2 to pre-pan well 3. Cover with a plate seal and incubate
for 40 min at room temperature on the vibrating platform
shaker.
Isolation of Artificial Binding Proteins
111
3.1 Affimer Selection
by Phage Display
Target proteins can be biotinylated using chemical conjugation
methods, such as EZ-Link NHS-Biotin (ThermoFisher Scientific,
catalog no. 20217), following the manufacturer’s instructions.
Chemical biotinylation may block the binding sites on target proteins. Alternatively, target protein sequence can be tagged with a
biotin acceptor peptide (BAP) sequence (GLNDIFEAQKIEWHE), which is enzymatically biotinylated when expressed in
E. coli in the presence of biotin ligase.
3.1.1 Panning Round 1
Day 1
1. Block the streptavidin-coated wells with 300 μl per well of
2Â Blocking Buffer. Prepare four wells per selection—three
wells for pre-panning the phage and one well to immobilize
the target. Cover the wells with an adhesive plate seal and
incubate overnight at 37
C.
2. Set up an overnight culture of ER2738 E. coli cells. Pick a single
colony into 5 ml of 2YT medium with 12 μg/ml tetracycline
and incubate overnight in an orbital incubator at 37
C,
230 rpm.
Day 2
3. Wash the blocked wells 3Â with 300 μl of PBST and replace
with 100 μl per well of 2Â Blocking Buffer.
4. Immobilization of biotinylated target and deselection target
(if using) (see Note 1). Add 1 μg of biotinylated target to the
selection well (see Note 2). If required, add 1 μg of biotinylated
deselection target into pre-pan wells 2 and 3. Proceed immediately to step 5.
5. Pre-pan 1. To the first pre-pan well, add 5 μl of Affimer phage
library (~10
12 colony-forming units). Cover with a plate seal
and incubate for 40 min at room temperature on a vibrating
platform shaker.
6. Pre-pan 2. Remove the blocking buffer/deselection target
from pre-pan well 2. If deselection target was used, wash the
well 6Â with 300 μl of PBST. Transfer the phage from pre-pan
well 1 to pre-pan well 2. Cover with a plate seal and incubate
for 40 min at room temperature on the vibrating platform
shaker.
7. Pre-pan 3. Remove the blocking buffer/deselection target
from pre-pan well 3. If deselection target was used, wash the
well 6Â with 300 μl of PBST. Transfer the phage from pre-pan
well 2 to pre-pan well 3. Cover with a plate seal and incubate
for 40 min at room temperature on the vibrating platform
shaker.
Isolation of Artificial Binding Proteins
111