Supplementary Figure S1 from Integrated Imaging Probe and Bispecific Antibody Development Enables In Vivo Targeting of Glypican-3–Expressing Hepatocellular Carcinoma

Supplementary Figure S1: Expression and Purification of TRAB Recombinant Protein. Synthesized TRAB gene fragments (IDT DNA, Coralville, IA), as specified in figure above (A), were inserted into a pTXB1 (New England Biolabs) vector using standard restriction enzyme based cloning techniques. Insertion of the TRAB sequence was verified by DNA sequencing using the T7 promoter as the sequencing primer. The TRAB-pTXB1 plasmids were then transformed into a competent E coli expression cell line (Origami 2 Cells, Novagen). Bacterial cell cultures were initially grown overnight in an air shaker (225 rpm) at 37°C in 3 mL of lysogeny broth (LB) media, eventually inoculated into scaled up 1 L LB media containing 50 mg/L of ampicillin. At optical density (OD) 600 nm = 0.6, Isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to a final concentration of 0.5 mM to induce T7 RNA polymerase-based expression. Cultures were allowed to express for 2 h at 37°C. Bacterial cultures were centrifugally pelleted at 10,000 g for 30 min, resuspended in 5 mL of column buffer (20 mM Na-HEPES, 0.5 M NaCl, 1 mM EDTA, pH 8.5) containing 0.75 g/L lysozyme and 50 mM phenylmethylsulfonyl fluoride. Cells were lysed by pulse sonication on ice. Cell lysate was centrifuged at 15,000 g for 30 min at 4°C. Supernatant was then loaded and incubated in Poly-Prep chromatography column (Bio-Rad, Hercules, CA) packed with Talon metal affinity resin (Clontech, Mountain View, CA). Supernatant was then allowed to pass through the column and resin beads were washed with multiple rounds of column buffer (0.1 M Tris-HCl, pH 8.5). Bound and washed TRAB protein (sequence shown in panel B) was then released from metal resin via the addition of 200 mM imidazole-containing elution buffer. Purification and concentration of the final product was performed using a 3 kDa molecular weight cutoff (MWCO) filter (Amicon Ultra, Milipore, Temecula, CA). The final product was analyzed on SDS-PAGE protein gel (Figure 2), with confirmation of the protein band at the expected molecular weight.