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Functional Protein Development

Mammalian cell recombinant protein expression platform

Focusing on recombinant proteins with functional activities

Figure 1.
Human SIRPα, hFC-His Tag (A) and CD47, mFc-His Tag(B) on SDS-PAGE under reducing condition

Figure 2.
ELISA plate pre-coated by 2ug/ml (100ul/well) Human CD47, mFc-His tagged protein (PME100008) can bind its native ligand Human SIRPα, hFc-His tagged protein (PME100009) in a linear range of 3.3-26.37ng/ml.

Figure 3.
ELISA plate pre-coated by 2ug/ml (100ul/well) Human CD47, mFc-His tagged protein (PME100008) can bind Magrolimab (Neutralising antibody clone Hu5F9-G4) in a linear range of 0.061-1.606ng/ml.

Validation of native receptor-ligand interaction

 


 

Lane 1: Human SIRPα, hFC-His tagged protein without freeze-thaw treatment

Lane 2: Human SIRPα, hFC-His tagged protein after one freeze-thaw cycle

Lane 3: Human SIRPα, hFC-His tagged protein after three freeze-thaw cycle

Lane 4: Human SIRPα, hFC-His tagged protein after five freeze-thaw cycle

Overall

Currently almost all the therapeutic antibody drugs target on extracellular domain of human membrane proteins or secreted protein molecules. To express and purify whole length membrane proteins in native confirmations sometimes are very challenging or impossible. The creation of ECD fusion protein is commonly used to approach to overcome this issue.

For druggable targets, DIMA will construct ECD fusion protein and use HEK293 mammalian expression system for production. This will not only ensure the authentic posttranslational modifications from mammalian cells, but also have a high chance of obtaining proteins in good solubility and correct folding under standard buffer system.

The flowchart of DIMA protein production platform

Key Advantages

  • Mammalian expression system: Clsoe to authentic posttranslational modification of native proteins
  • Serum-free culture system: Low endotoxin evel and less host cell protein contamination
  • ECD fusion protein will improve solubility and stability
  • Codon optimisation can further improve the ptrotein expression level

Key applications of DIMA mammalian cell expressed proteins

  1. Native immunogens for therapeutic antibody drug development
  2. Native ligand and receptor identification
  3. Therapeutic drug in vitro functional test
  4. Cell based assays
  5. Biophysical evaluation of therapeutic drug