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MEXi is optimized for high throughput mammalian expression and efficient purification

What are the problems of common mammalian expression systems?

Problems you might face if you use a mammalian expression system for protein production:

  • Up- and downstream processes are usually developed independently, so a fit of both is usually accompanied by intensive optimization
  • His-tag purification from eukaryotic cells usually results in high impurities due to interference of substances in the medium and other intrinisc proteins containing histidine that interfere unspecifically with the purification matrix
  • Cultivation media contain components which interfere with downstream process
  • Purification buffers contain substances, which should be avoided for downstream applications (e.g. high imidazole concentrations)

A solution might be:

  • For His-tag purification: do time and cost intensive dialysis procedures and buffer exchanges
  • For Strep-tag® purification: block Biotin in the cultivation medium with the economic BioLock solution
How does MEXi meet the needs of a mammalian expression system?

What are the needs?

  • High throughput of different recombinant proteins for screenings etc.
  • Efficient purification (cost and time saving)

How does MEXi meet those needs?

All components of the MEXi system are optimized for an efficient protein production and purification using Strep-tag®:

  • Growth of MEXi293E cells is independent of changes during cultivation (e.g. high density)
  • Transient expression of the recombinant protein in an oriP/EBNA-1 dependent manner, which ensures high expression levels
  • The transfection protocol is optimized for the time and cost saving PEI transfection
  • The MEXi293E cells have a stable transfection efficiency throughout a wide range of passages
  • pDSG expression vector series is IP free
  • pDSG vectors are small and equipped with oriP for episomal replication and high plasmid yields in the cells
  • Low biotin content in the expression medium for optimal Strep-tag® purification and easy blockage of biotin using BioLock
  • Purification via Strep-tag® usually results in a protein purity < 95 %
  • Strep-tag® is suited for a variety of downstream applications such as high affinity applications

Learn more about MEXi

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

 

 

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
Expression of a mAb with MEXi

 

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
Expression of a customer protein (POI) using MEXi

 

 

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.