Hitrap columns

Luria broth and isopropyl β-d-1-thiogalactopyranoside (IPTG) were purchased from Research Products
International as well as reagents for Terrific Broth (TB) medium.
HEPES, triethanolamine, ammonium acetate, UTP, glucose 6-phosphate,
sodium pyruvate, and NAD⁺ were purchased from Sigma-Aldrich,
in addition to the enzymes pyrophosphatase, lactate dehydrogenase,
and phosphoglucomutase. The 1 and 5 mL HiTrap columns and 5 mL Nickel
NTA HisTrap columns and Vivaspin 500s and Vivaspin 20 10 kDa filters
were purchased from GE Healthcare. The PA1 Dionex Carbopac guard columns
(4 × 50 mm) and Dionex Carbopac PA1 analytical columns (4 ×
250 mm) were purchased from Thermo-Scientific. The 0.2 μm 47
mm nylon filters for the PA1 columns were purchased through VWR from
the PALL Corporation. Sodium dithionite was purchased from Thermo-Fischer.
UDP-d-glucuronate was obtained from Biosynth Carbosynth.
Oxygen-18 labeled water (97%) was purchased from Medical Isotopes
Inc. UDP-β-l-arabinofuranoside (7) was
chemically synthesized by the modification of previously published
procedures as outlined in the Supporting Information.

Cytosols obtained from 500 mL cultures were used for purification using HiTrap columns (GE Healthcare). Samples were first injected on Q-Sepharose cartridges (5 mL) equilibrated in buffer A at 1 mL/min, washed with 5 volumes of this buffer (until A₂₈₀ = 0), and eluted with a gradient (10 column volumes) from 0 to 1.5 M KCl in buffer A. Fractions (2 mL) were collected for activity measurements and those exhibiting AdoMet synthesis were pooled. Active peaks were then loaded on phenyl Sepharose cartridges (5 mL) equilibrated in buffer A containing 300 mM KCl at 1 mL/min, washed with 5 volumes of this buffer, and eluted with 2 bed volumes of buffer A containing 50% (v/v) DMSO. Fractions (4 mL) were collected and assayed for AdoMet synthesis activity. The active fractions were pooled and extensively dialyzed against buffer A for DMSO elimination. Dialyzed proteins were concentrated through YM-30 membranes (AMICON Corp., Beverly, MA, USA) before use. Samples of each purification step were loaded on 10% SDS-PAGE gels and stained with Coomassie Blue to assess purity.

As previously described^{9 (link)}: E. coli Bl21 star™ cells were heat shock transformed with plasmid pET-28b(+)-BaSP-Q345F. Overnight cultures of the transformed host in LB-medium containing 50 mg/L kanamycin sulfate were grown and 1.8 mL was used to inoculate 250 mL of LB-medium (50 mg/L kanamycin sulfate). The cultures were incubated at 37 °C and 180 rpm until they reached an OD600 of 0.6, at which point the temperature was adjusted to 19 °C and IPTG was added to a final concentration of 0.5 mm. The cells were grown for additional 18 hours after which they were harvested by centrifugation (4000 g for 10 min). The sediment was resuspended in lysis buffer (60 mm phosphate, 250 mm NaCl, 11 mm imidazol). Cells were lysed using a sonicator and centrifuged at 17000 g for 10 min at 4 °C. The lysate was loaded onto 0.5 mL Ni-NTA columns equilibrated with lysis buffer and incubated at 4 °C and slow rotation for a minimum of 2 hours. The column was washed with 2.5 mL of lysis buffer and the protein was eluted with 1.5 mL of elution buffer (60 mm phosphate, 250 mm NaCl, 230 mm imidazol). The buffer was exchanged to 20 mm MOPS-NaOH-buffer (pH = 7) using 5 mL Hi-Trap columns from GE Healthcare.

To produce anti-cancer/anti-CD3 BsAbs, the BsAb plasmids (30 μg) were transfected into Expi293 cells with ExpiFectamine (Thermo Fisher Scientific, Waltham, MA, USA). The BsAbs were harvested 5 days after the plasmid transfection. Pure anti-PSMA Fab/anti-CD3 scFv, anti-PSMA scFv/anti-CD3 Fab, and anti-PSMA scFv/anti-CD3 scFv were obtained by using HiTrap columns (GE Healthcare, Munich, Germany). Pure anti-PSMA hole /anti-CD3 konb were obtained by using HiTrap and HA columns (GE Healthcare). The concentrations of purified BsAbs were determined by Pierce™ BCA protein assay kit (Thermo Fisher Scientific). The purity levels of BsAbs were analyzed by non-reducing or reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

CCP2 peptides (2 mg) (corresponding to the patent-protected peptides used in the commercially available Immunoscan CCPlus® assay from Euro-Diagnostica AB) were coupled to 1 mL HiTrap columns (GE Healthcare). Recommended flow rate and backpressure specifications were followed (flow rate: 1 mL/min, maximum backpressure: 0.3 MPa). Performance of the columns was typically around 1 mg anti-CCP2 IgG/column.