8. Day 2: Collect 2 mL fractions. Analyze the UV trace, and pool
the peak fractions.
9. Perform SDS-PAGE to check the purity.
10. Measure the protein concentration of each fraction using a
NanoDrop spectrophotometer to estimate the protein yield.
11. Use an aliquot to evaluate the biophysical and/or functional
properties of the purified mCherry-engineered Fab fragment
(Fig. 5 as a typical example). Ideally, binding assays, such as
SPR, ITC, or ELISA, should provide valuable information.
12. Concentrate the remaining purified fluorescent Fab fragment
using an Amicon Ultra-15 (MWCO 30 K) centrifugal concentrator to an appropriate protein concentration.
13. Re-measure the protein concentration of each fraction using a
NanoDrop spectrophotometer.
14. Prepare 100 μL aliquots and flash-freeze with liquid nitrogen.
15. Store at À80
C.
3.2.4 Purification
of Tag-Free Fab Fragments
1. Day 1: (IMAC) After collecting the Sf9 culture supernatant,
perform the same procedures as steps 1–5 in Subheading
3.2.3.
2. (TEV cleavage and dialysis) Add an appropriate amount (typically at a protease to target protein ratio of 1:100 (w/w) or
3 mg) of TEV-His 6 to the elution fraction.
3. Transfer the mixture into dialysis tubing, and dialyze overnight
at 4
C against 2 L of TBS.
4. Day 2: (Reverse IMAC) Equilibrate a 5-mL HisTrap HP column with 25 mL of buffer C.
5. Remove the dialysate from the tubing and inject it into the
column using a 10-mL syringe.
ä
Fig. 5 (continued) caused by boiling before SDS-PAGE as described in [18]. (d) SEC profile of the mCherryengineered certolizumab Fab-TNFα complex in the presence of excess engineered Fab on a Superdex200
10/300GL column (blue line). As a reference, TNFα alone was separated on the same column (green line).
Elution volumes of protein standards are indicated at the top. Peak Fab*-T engineered certolizumab Fab-TNFα
trimer complex, peak T free TNFα trimer, and peak Fab* free engineered certolizumab Fab. (e) FSEC profile.
The fractions collected in (d) were subjected to mCherry fluorescence measurement. Red line: mixture of the
engineered certolizumab Fab and TNFα trimer; pink line: TNFα trimer alone. (f) SDS-PAGE analysis of the
corresponding peak fractions in (d). (g) In gel fluorescence analysis of mCherry-engineered certolizumab Fab.
The same peak fractions as in (f) were added 1:1 with buffer containing 50 mM Tris-HCl (pH 7.6), 50 mM DTT,
5% glycerol, 5% SDS, 5 mM EDTA, and 0.02% bromophenol blue. Without boiling the sample, electrophoresis
was performed on a Novex WedgeWell 10% Tris-glycine gel (Thermo Fisher Scientific) at 100 V for 130 min.
For mCherry, protein bands were observed on a LED transilluminator. The theoretical molecular masses of the
red fluorescent certolizumab Fab and TNFα trimer are 81.2 kDa and 51.9 kDa (17.3 kDa protomer Â3),
respectively
Production of Recombinant Antibody Fragments via the iRAT system
99
the peak fractions.
9. Perform SDS-PAGE to check the purity.
10. Measure the protein concentration of each fraction using a
NanoDrop spectrophotometer to estimate the protein yield.
11. Use an aliquot to evaluate the biophysical and/or functional
properties of the purified mCherry-engineered Fab fragment
(Fig. 5 as a typical example). Ideally, binding assays, such as
SPR, ITC, or ELISA, should provide valuable information.
12. Concentrate the remaining purified fluorescent Fab fragment
using an Amicon Ultra-15 (MWCO 30 K) centrifugal concentrator to an appropriate protein concentration.
13. Re-measure the protein concentration of each fraction using a
NanoDrop spectrophotometer.
14. Prepare 100 μL aliquots and flash-freeze with liquid nitrogen.
15. Store at À80
C.
3.2.4 Purification
of Tag-Free Fab Fragments
1. Day 1: (IMAC) After collecting the Sf9 culture supernatant,
perform the same procedures as steps 1–5 in Subheading
3.2.3.
2. (TEV cleavage and dialysis) Add an appropriate amount (typically at a protease to target protein ratio of 1:100 (w/w) or
3 mg) of TEV-His 6 to the elution fraction.
3. Transfer the mixture into dialysis tubing, and dialyze overnight
at 4
C against 2 L of TBS.
4. Day 2: (Reverse IMAC) Equilibrate a 5-mL HisTrap HP column with 25 mL of buffer C.
5. Remove the dialysate from the tubing and inject it into the
column using a 10-mL syringe.
ä
Fig. 5 (continued) caused by boiling before SDS-PAGE as described in [18]. (d) SEC profile of the mCherryengineered certolizumab Fab-TNFα complex in the presence of excess engineered Fab on a Superdex200
10/300GL column (blue line). As a reference, TNFα alone was separated on the same column (green line).
Elution volumes of protein standards are indicated at the top. Peak Fab*-T engineered certolizumab Fab-TNFα
trimer complex, peak T free TNFα trimer, and peak Fab* free engineered certolizumab Fab. (e) FSEC profile.
The fractions collected in (d) were subjected to mCherry fluorescence measurement. Red line: mixture of the
engineered certolizumab Fab and TNFα trimer; pink line: TNFα trimer alone. (f) SDS-PAGE analysis of the
corresponding peak fractions in (d). (g) In gel fluorescence analysis of mCherry-engineered certolizumab Fab.
The same peak fractions as in (f) were added 1:1 with buffer containing 50 mM Tris-HCl (pH 7.6), 50 mM DTT,
5% glycerol, 5% SDS, 5 mM EDTA, and 0.02% bromophenol blue. Without boiling the sample, electrophoresis
was performed on a Novex WedgeWell 10% Tris-glycine gel (Thermo Fisher Scientific) at 100 V for 130 min.
For mCherry, protein bands were observed on a LED transilluminator. The theoretical molecular masses of the
red fluorescent certolizumab Fab and TNFα trimer are 81.2 kDa and 51.9 kDa (17.3 kDa protomer Â3),
respectively
Production of Recombinant Antibody Fragments via the iRAT system
99