Kta pure system

Milk was centrifuged at 2500 rpm for 20 min at 4 °C to remove the fat. The skimmed milk was acidified to pH 4.6 to precipitate casein and centrifuged at 100,000 × g at 20 °C for 1 h. The purification procedure was performed using an ÄKTA pure system (GE Healthcare, Uppsala, Sweden). First, after equilibration in a column with equilibration buffer (20 mM phosphate buffer (PB), pH 8.2), samples were loaded into a HiScreen SP Sepharose FF column (GE Healthcare, Uppsala, Sweden; 4.7 mL) and the protein was eluted with a linear gradient of 0–1 M NaCl in 20 mM PB, pH 8.2. Then, an Ultracel-30 membrane (Millipore Corporation, Billerica, MA, USA) was used to concentrate the fractions containing rhLZ on the ÄKTA Crossflow automated cross flow filtration system (GE Healthcare, Uppsala, Sweden) After purification, the rhLZ was exchanged by 20 mM PB and the quantity and quality of the purified rhLZ was detected by SDS-PAGE.

DFE was purified on an ÄKTApure system (GE Healthcare, Uppsala, Sweden) fitted with an XK-26 column containing 53 ml of chelating Sepharose fast-flow-immobilized metal-ion affinity chromatography (IMAC) resin loaded with nickel ions and pre-conditioned with extraction buffer lacking sodium bisulfite. After loading the clarified extract, unbound proteins were washed through with 10 column volumes of the same buffer, and bound proteins were then eluted in the same buffer supplemented with 300 mM imidazole at a flow rate of 50 cm h⁻¹. The protein and nucleic acid concentrations were monitored at 280 and 260 nm, respectively.

VH and VL domain fragments from selected anti-EGFR scFvs were subcloned into the expression vector pMH3 to generate the full-length human IgG1 (Cκ) format. The cetuximab variants Ctx-Y104X constructs were generated by site-directed mutagenesis. Primers used in this experiment were listed in Supplementary Table 9. For the control antibody, the VH and VL domains of another anti-EGFR antibody, panitumumab, were cloned into the expression vector pMH3 to generate full-length human IgG2/κ. Recombinant antibodies were produced in HEK293F cells through transient transfection. Antibodies were purified from culture supernatants using a HiTrap Protein A column (GE Healthcare, PA, USA) in an ÄKTA pure system (GE Healthcare, PA, USA) and were dialyzed against PBS (pH 7.4). The purity and homogeneity of cetuximab variants were analyzed by size-exclusion chromatography-high performance liquid chromatography (SEC-HPLC) and date was acquired with the Agilent 1200 & EZChrom Elite software (Agilent Technologies, Palo Alto, CA, USA). The stability of cetuximab variants were analyzed by dynamic light scattering (DLS). LitesizerTM 500 (Anton paar, USA) was used for DLS data acquisition.

The recombinant plasmids were linearized with endonuclease Sac I and transformed into P. pastoris X-33 cells by a Gene Pulser Xcell™ Electroporation System (Bio-Rad, Hercules, CA, USA) working at 2,000 V and 5 ms. The transformants were screened on YPDS plates (10 g/L yeast extract, 20 g/L peptone, 20 g/L dextrose, 1 mol/L sorbitol, and 20 g/L agar) containing 100 μg/mL of Zeocin for 2–3 days.
Recombinant yeast culture and protein expression were conducted according to the method of Cao et al.^{13 (link)}. The supernatant of the culture was collected by centrifugation at 5,000 rpm for 5 min, followed by precipitation with ammonium sulphate of 75% saturation degree on ice. The precipitate was collected by centrifugation at 12,000 rpm for 10 min and dissolved in 6 mL of 50 mM HAc-NaAc buffer (pH 5.3), then dialysed in 50 mM HAc-NaAc buffer (pH 5.3) in order to remove ammonium sulphate. After dialysis, the crude enzyme was purified via strong anion exchange column (UNOsphere Q, Bio-Rad) by gradient elution with 0–1 M NaCl (ÄKTA™ pure system, GE Healthcare). The active fractions were pooled and stored at 4 °C for further analysis. Protein concentration was determined with a Pierce^TM BCA Protein Assay Kit (Thermo, USA).