MEXi is optimized for mammalian expression and efficient purification
Rapid high‑yield expression and purification of fully post‑translationally modified recombinant clusterin and mutants
Satapathy et al. demonstrate the utility of IBA’s MEXi system for mammalian expression of fully functional and even heavily post-translationally modified proteins, enabling the comprehensive analysis of structure-function relationships of difficult-to-express, low abundant, or even secreted targets. In doing so, the researchers exploited the flexible fusion options of the Twin-Strep-tag® to wild type and mutant clusterins, secreted chaperones involved in the capture of toxic aggregating proteins in extracellular body fluids, which exhibit highly complex modifications and tend to form heterogeneous high-molecular-weight aggregates. After protein production using MEXi-293E cells in combination with our specially prepared transfection and culture media, they were able to obtain highly pure and functional clusterins upon elution from the Strep-Tactin®XT Superflow® high capacity cartridge. As compared to prior expression and purification strategies, the researchers report a substantial increase in yield from former 4.5 mg/L to current 30 mg/L directly from human plasma cultures, deploying a straightforward single-step chromatography protocol. Further, they made use of the integrated Strep-tag® toolbox by applying our StrepMAB-Classic antibody and Strep-Tactin®HRP conjugate for detection by cell staining as well as western blotting. In summary, Satapathy and colleagues showcase an efficient workflow for minimization of time, labor and resources in expression, purification and adjustable downstream analysis of demanding proteins and their respective mutants.
Read the full paper here.
Further examples of MEXi system applications:
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.
Comparison of IBA's mammalian expression system MEXi with a competitor system:
||MEXi||Competitive Expression System (CES)
|SEAP yield (mg/L)||145||140|
|medium||MEXi media||Freestyle F17|
|medium list price [EUR]||79||163|
seeding cell density
(ratio compared to CES)
costs* for 1L expression
(ratio compared to CES)
|*costs implement Medium, Cells, DNA and working time|
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