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Purification of SEAP fused with Twin-Strep-tag® after expression using the MEXi system

 

 

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.

Purification of rat mAb fused with Twin-Strep-tag® after expression using the MEXi system

 

The obtained protein yield of the rat antibody was 96 mg/l represented by heavy (HC) and light chain (LC).
 
A rat monoclonal antibody (mAb) was cloned into the pDSG-IBA102 vector in order to fuse the heavy chain (HC) C-terminally with the Twin-Strep-tag® and the BM40 secretion signal. The transfection of MEXi 293E cells was performed in MEXi-TM transfection medium with 25 kDa PEI in a 250 ml culture volume. Afterwards the cells were incubated for 4 hours at 37°C and then 250ml MEXi-CM culture medium was added. When the cells reach a cell density of 3x106 cells/ml. At this concentration the culture was shifted to 32°C and incubated until day 10.

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.

Purification of POI fused with Twin-Strep-tag® after expression using the MEXi system

 

 

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.