10. Elute the polyprotein of interest from the column by adding
three CVs (15 mL) of buffer B and collect the eluate into a
50-mL Falcon tube.
11. Measure the protein concentration of each fraction using a
NanoDrop spectrophotometer.
12. (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.
13. Transfer the mixture into dialysis tubing, and dialyze overnight
at 4
C against 2 L of TBS.
14. Day 3: (Reverse IMAC) Equilibrate a 5 mL HisTrap HP column with 25 mL of buffer C.
15. Remove the dialysate from the tubing and inject it into the
column using a 10 mL syringe.
16. Collect the flow-through fraction (FT).
17. Wash the column three times, each wash containing 1 CV
(5 mL) of buffer C and collect as W1, W2, and W3 fractions,
respectively.
18. Elute the bound material (excised iRAT-MBP segment, TEV-His 6 , and contaminating proteins) from the column by adding
three CVs (15 mL) of buffer D and collect as E fraction.
19. Measure the protein concentration of each fraction using a
NanoDrop spectrophotometer.
20. Mix the FT and W1 fractions and concentrate the protein using
a Amicon Ultra-15 (MWCO 10 K) centrifugal concentrator to
a final volume of 5 mL.
21. (Size-exclusion chromatography (SEC)) Inject the 5 mL of
concentrated protein sample onto a HiLoad16/60 Superdex200 column pre-equilibrated with TBS at a flow rate of
1 mL/min.
22. Collect 2 mL fractions. Analyze the UV trace, and pool the
peak fractions.
23. Perform SDS-PAGE to check the purity.
24. Measure the protein concentration of each fraction using a
NanoDrop spectrophotometer to estimate the protein yield.
25. Use an aliquot to evaluate the biophysical and/or functional
proprieties of the purified tag-free Fv fragment (Fig. 3 as a
typical example). Ideally, binding assays, such as surface plasmon resonance (SPR), isothermal titration calorimetry (ITC),
and ELISA, should provide valuable information.
26. Concentrate the remaining purified Fv fragment using an Amicon Ultra-15 (MWCO 10 K) centrifugal concentrator to an
appropriate protein concentration (e.g., 10 mg/mL).
Production of Recombinant Antibody Fragments via the iRAT system
89
three CVs (15 mL) of buffer B and collect the eluate into a
50-mL Falcon tube.
11. Measure the protein concentration of each fraction using a
NanoDrop spectrophotometer.
12. (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.
13. Transfer the mixture into dialysis tubing, and dialyze overnight
at 4
C against 2 L of TBS.
14. Day 3: (Reverse IMAC) Equilibrate a 5 mL HisTrap HP column with 25 mL of buffer C.
15. Remove the dialysate from the tubing and inject it into the
column using a 10 mL syringe.
16. Collect the flow-through fraction (FT).
17. Wash the column three times, each wash containing 1 CV
(5 mL) of buffer C and collect as W1, W2, and W3 fractions,
respectively.
18. Elute the bound material (excised iRAT-MBP segment, TEV-His 6 , and contaminating proteins) from the column by adding
three CVs (15 mL) of buffer D and collect as E fraction.
19. Measure the protein concentration of each fraction using a
NanoDrop spectrophotometer.
20. Mix the FT and W1 fractions and concentrate the protein using
a Amicon Ultra-15 (MWCO 10 K) centrifugal concentrator to
a final volume of 5 mL.
21. (Size-exclusion chromatography (SEC)) Inject the 5 mL of
concentrated protein sample onto a HiLoad16/60 Superdex200 column pre-equilibrated with TBS at a flow rate of
1 mL/min.
22. Collect 2 mL fractions. Analyze the UV trace, and pool the
peak fractions.
23. Perform SDS-PAGE to check the purity.
24. Measure the protein concentration of each fraction using a
NanoDrop spectrophotometer to estimate the protein yield.
25. Use an aliquot to evaluate the biophysical and/or functional
proprieties of the purified tag-free Fv fragment (Fig. 3 as a
typical example). Ideally, binding assays, such as surface plasmon resonance (SPR), isothermal titration calorimetry (ITC),
and ELISA, should provide valuable information.
26. Concentrate the remaining purified Fv fragment using an Amicon Ultra-15 (MWCO 10 K) centrifugal concentrator to an
appropriate protein concentration (e.g., 10 mg/mL).
Production of Recombinant Antibody Fragments via the iRAT system
89