Transient Protein Production

  • Benefits of the Nucleofector™ Technology

    Transient protein expression in mammalian and insect cell lines has gained increasing relevance as it enables fast and flexible production of high-quality eukaryotic protein. Milligram amounts of protein can be produced within several days, meaning a significant shortening of process time in comparison to protein production from stable clones where generation of producer cells and production of protein can take up to several months.  

    Transient protein expression & production offers:

    • Fast production of small-scale protein amounts
    • Low costs for cell maintenance  

    In the past gene transfer into serum-free cultivated mammalian and insect cells suitable for protein production was hampered by inefficient transfection methods. The Nucleofector™ Technology represents the ideal tool for gene transfer even into difficult-to-transfect cell lines like suspension CHO cells. It has now been successfully tested and validated for its use in transient protein production.  


    Benefits of the Nucleofector™ Technology for transient protein production:

    • Milligram amounts of protein in just a few days – High transient protein expression yields
    • Applicable for your favourite cell clone – Suitable for any individual cell line and clone, e.g. suspension CHO, suspension HEK293, BHK-21, NSO, S2 and many other cells
    • Fast onset of expression – Direct transfer of DNA into the nucleus
    • Single to 96-well format – Accelerating and standardizing your research
    • Simple up-scaling of experiments –  From 106 to 107 cells per single Nucleofection™ or up to 2x108 cells per 96-well Nucleocuvette™ Plate
    • Flexibility in choice of cultivation media – Applicable even under serum-free conditions
  • Nucleofection™ is Superior to Lipofection

    In a comparative study the Nucleofector™ Technology and the lipofection reagent Lipofectamine™ 2000 (Invitrogen) have been tested in regard to their suitability for transient protein production. Nucleofection™ achieves 10-30 times higher expression rates in CHO cells compared to cells transfected with lipofection. A similar ratio has been determined for both the specific and the volumetric productivities.  


    sCHO - hSEAP productivity
    Nucleofection™ is superior to lipofection for transient protein production. Two different suspension CHO cell clones (sCHO clone 1 and sCHO clone 2) have been transfected with a plasmid encoding the human secreted alkaline phosphatase (pDRIVE-hSEAP) either using Lipofectamine™ 2000 or Nucleofection™.   For lipofection, 58 µg plasmid (sCHO clone 1) or 116 µg plasmid (sCHO clone 2) were transfected with 290 µl or 580 µl lipd reagent, resepectively. For Nucleofection, 25 mg plasmid were transfected using Cell Line Nucleofector™ V and program U-024. Post transfection, 5 x 107 cells (pooled from 5 single samples, 1 x 107 cells each) were cultivated as batches in spinner flasks containing 50 ml culture medium for 5 days under non-regulated conditions. Specific productivities (µUnits/cell/day) of hSEAP referred to a cell density of 106 cells/ml seeded after the respective transfection.

    Suspension 293 cells (DSMZ, adapted from adherent cell clone) have also been tested successfully. 5 x 107 (= 5 samples) cells have been transfected with 25 μg pDrivehSEAP using Cell Line Nucleofector™ Kit V in combination with program X-001. A specific productivity of 2.4 µU/c/d and a volumetric productivity of 10.5 U/ml could be obtained. These cells were not transfectable using lipofection.  

     

  • Antibody Production using Nucleofection™

    The Nucleofector™ Technology was also tested successfully for its suitability to produce functional proteins. 2 mg of a human IgG1 antibody was produced within four days.  


    sCHO - antibody production
    Antibody production after Nucleofection™ of CHO suspension cells. 1 x 108 sCHO cells (clone 2) were transfected using Cell Line Nucleofector™ Kit V in combination with program U-24/U-024. Cells were co-transfected with plasmids encoding either the DNA sequence of the light chain or the heavy chain of a human IgG1 antibody. Post Nucleofection™ cells were cultivated for 5 days in serum-free medium. Cells reached a specific productivity of 5.5 pg/c/d and a volumetric productivity of 39 μg/ml. Within 4-5 days 2 mg of the antibody could be produced (determined by ELISA). The integrity of the produced antibody was verified via Western blotting.

  • Nucleofection™ of Insect Cells

    For many research protein applications insect cells provide the ideal expression system, as they combine the ability to produce proteins containing eukaryotic post-translational modifications with both high productivity and simple cultivation needs.  


    Benefits of the Nucleofector™ Technology for protein production in insect cells: 

    • Up to 82% transfection efficiency and 79% cell viability for most common insect cell lines incl. Sf9 and S2 cells
    • Transient protein expression with high yields
    • Time-savings in generation of baculovirus due to excellent efficiency of bacmid delivery
    • 96-well Nucleofection™ Platform available for fast and standardized screening of bacmid or other vector constructs
    • Easy-to-follow protocols with cell culture details  


    Transient protein production in S2 cells
    Excellent protein production rates of S2 insect cells 24 hours post Nucleofection™. S2 cells were transfected with insect expression vector encoding the LacZ/β-gal gene. Each bar represents the average β-gal enzyme activity from six replicates 24 h post-transfection, demonstrating the excellent protein production rates achieved by Nucleofection™ already after 24 h. β-gal enzyme activities were at similar levels 48 h after transfection (data not shown).