Protein Protocols Protein Protocols

Single-Chain Antibodies 1041
XL1-Blue, combine the culture supernatant and the soluble periplasmic extract, clarify by
additional centrifugation (30,000g, 4°C, 60 min), and pass through a glass filter of pore
size 10–16 µm and then through a filter of pore size of 0.2 µm.
3. Either reduce the vol 10× by concentrating with Amicon ultrafiltration cell and YM 10
membrane (see Note 9) or concentrate the bispecific recombinant product by ammonium
sulfate precipitation (see Note 10). In the latter case, place the beaker with the culture
supernatant and the soluble periplasmic extract on a magnetic stirrer. Slowly add ammonium
sulfate powder to a final concentration 70% of saturation (472 g/L of solution).
Continue stirring for at least another 2 h at 4°C.
4. Collect the protein precipitate by centrifugation (30,000g, 4°C, 30 min) and dissolve it in
1/10 of the initial volume of 50 mM Tris-HCl, 1 M NaCl, pH 7.0.
5. Thoroughly dialyze the concentrated protein against 50 mM Tris-HCl, 1 M NaCl, pH 7.0
at 4°C. Clarify the dialyzed material by centrifugation (30,000g, 4°C, 60 min).
6. For IMAC, prepare a column of chelating Sepharose (1–2 mL of resin/L of flask culture),
wash with 5 bed volumes of water. Charge the column with Cu 2+ by loading 0.7 bed
volume of 0.1 M CuSO 4 (see Note 11), wash the excess of ions with 10 bed volumes of
water, and equilibrate with 3 volumes of 50 mM Tris-HCl, 1 M NaCl, pH 7.0 (see Note 12).
7. Pass the soluble periplasmic proteins over a chelating Sepharose column either by gravity
flow or using a peristaltic pump. Wash the column with 10 bed volumes of start buffer
(50 mM Tris-HCl, 1 M NaCl, pH 7.0) followed by start buffer containing 50 mM imidazole
(see Note 13) until the absorbance (280 nm) of the effluent is minimal (20–30 column
volumes). Perform all chromatography steps at 4°C.
8. Elute bound antibody fragments with a start buffer containing 250 mM imidazole (see
Note 14).
9. Analyze the purity of eluted material by SDS-PAGE (35).
3.4. Final Purification of SCA Fragments and Analysis
of Molecular Forms
1. If the IMAC yields in homogeneous preparation of recombinant protein according to
reducing SDS-PAGE, go to step 6 (see Note 15). Otherwise, calculate the isoelectric point
(pI) of your SCA on the basis of amino acid composition of antibody fragment (see Note 16).
2. Subject the protein material eluted from the IMAC column to buffer exchange either for
50 mM imidazole-HCl, pH 6.0–7.0, or 20 mM Tris-HCl, pH 8.0–8.5, using prepacked
PD-10 columns (see Note 17). Remove the turbidity of protein solution by centrifugation
(30,000g, 4°C, 30 min).
3. Load the protein solution either on a Mono Q or Mono S column equilibrated either with
20 mM Tris-HCl, pH 8.0–8.5, or 50 mM imidazole-HCl, pH 6.0–7.0, respectively. Wash
the column with a least 10 volumes of the start buffer.
4. Elute the bound material using a linear 0–1 M NaCl gradient in the start buffer; collect
1-mL fractions.
5. Perform sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis
of eluted fractions.
6. Pool the fractions containing pure recombinant antibodies. Determine the protein concentration
(see Note 18).
7. Perform a buffer exchange for PBSI, pH 7.0–7.4, (see Note 19) and concentrate the purified
antibody preparations up to 1.0–2.0 mg/mL using Ultrafree-15 centrifugal filter units.
8. Equilibrate a Superdex column with PBSI buffer; calibrate the column using high and low
molecular weight gel filtration calibration kits (see Note 20).