Akta pure 25 m

The samples of protein DNA mixture were analyzed by AKTA Pure 25 M (GE, Boston, MA, USA) using EzLoad 16/60 Chromdex 200 pg (Bestchrom, Shanghai, China) at 25 °C and buffer (20 mM HEPES, pH 7.5, 200 mM NaCl, 50 mM CaCl₂, 10 mM MgCl₂ and 10 mM KCl) flowing at 0.2 mL/min after MtrA (10 µM) associated with dsDNA fragments (20 µM) in the same buffer and incubated for 30 min at 25 °C. The sequences of the DNA fragment used for size exclusion chromatography were synthesized by Tsingke Biotechnology (Beijing, China) and are listed in Table 1.

For fluorescence-detection size exclusion chromatography (FSEC), 10 mL of cells were transfected with the desired constructs, as described above. After 48 h, 5 mL of cells were centrifuged at 4,000 g for 10 min at 4 °C. The cell pellets were resuspended in 400 μl solubilization buffer and incubated with 2% DDM and 0.4% CHS at 4 °C for 1 h. The lysates were centrifuged at 15,000g for 30 min at 4 °C. The supernatants were filtered using a centrifuge tube filter (Corning Costar Spin-X) and diluted 2-fold with FSEC buffer containing 50 mM Tris-Cl pH 8.0, 200 mM NaCl, 0.03% DDM, 0.006% CHS, 1 mM TCEP, and 1 mM l-aspartate. Fifty microlitre samples were injected into a Superose 6 Increase 10/300 column preequilibrated with FSEC buffer. GFP fluorescence was monitored by a fluorescence detector (Shimadzu RF-20A) in-line with AKTA pure 25 M (GE Healthcare).

Eukaryotic expression system was used to purify human EphA2 and HCMV gH/gL. Briefly, expression plasmid was mixed with Polyethyleneimine Linear (PEI) MW40000 (40816ES, Yeasen, China) at a molar ratio of 1:3 in 293F medium (UP1000, Union, China), and the mixture was transfected to Expi293F (A14527, ThermoFisher, Massachusetts). Then the cells were cultured at 37°C with 5% CO2 and 120 rpm shaking for 6 days, and the supernatant was harvested by centrifuge, filtered with 0.45 um filtering membrane, and applied to gravity column loaded with Ni Sepharose Excel (17371202, Cytiva, Washington, DC). The column was washed by PBS with 30 mM imidazole and eluted by PBS with 500 mM imidazole. The elution was concentrated by ultracentrifuge and further purified by size exclusion chromatography (SEC) using Superdex 200 increase 10/300GL (28990944, Cytiva, Washington, DC) on an AKTA Pure25M (GE healthcare, Massachusetts).