MEXi expression system is optimized for high expression yields
Obtained protein yield of SEAP after purification: 143 mg/l.
Secreted alkaline phosphatase (SEAP) was fused with a C-terminal Twin-Strep-tag® (TST) and the BM40 secretion signal via cloning into pDSG-IBA102. MEXi 293E cells were transfected in MEXi-TM transfection medium with 25 kDa PEI (polyethylenimine) in 17 ml culture volume. Afterwards the cells were first incubated for 4 hours at 37°C before MEXi-CM culture medium was added. The cells were kept at 37°C for 7 days in order to obtain high protein yields. For purification, the cells were pelleted and the supernatant, containing the SEAP protein, was harvested. SEAP protein was finally purified using a Gravity flow Strep-Tactin® Superflow® high capacity column.
In order to divide the cells from the supernatant the cell suspension was centrifuged according to the MEXi manual. The supernatant was used for protein purification via a Gravity flow Strep-Tactin® Superflow® high capacity column. WET FRED was used to facilitate loading of the large supernatant volume onto the column.
Obtained protein yield of POI after purification: 318 mg/l.
The protein of interest (POI) comprises a C-terminal Twin-Strep-tag® and the BM40 secretion signal. It was cloned into pDSG-IBA102. 1050 ml of MEXi-TM medium was inoculated with MEXi 293E cells. Subsequently, plasmid DNA was added followed by addition of 25 kDa linear PEI. The cells were incubated for 4 hours in MEXi-TM medium at 37°C and 5 % CO2 in an orbital shaking incubator. Cells were diluted to 7.5 x 105 cells/ml by the addition of one volume MEXi-CM culture medium and kept at 37°C for 7 days. Afterwards, they were pelleted and the supernatant, containing POI, was harvested. POI was finally purified using Strep-Tactin® Superflow® XT. The figure shows the elution fractions (E1-E3). A dilution of 1:10 was prepared for E1 and E2 before application to SDS-PAGE.