The production of recombinant proteins with complex post-translational modifications is crucial for studying their structure and function. One effective method for achieving high protein yields is through the transient transfection of mammalian cell lines, particularly the HEK293F and HEK293S cell lines, using a mammalian expression vector. This protocol demonstrates the applicability of the system by expressing three glycoproteins, including human FcγRIIIa and rat α2-6 sialyltransferase (ST6GalI), with yields ranging from 95-120 mg per liter of culture. These proteins, along with unmodified human immunoglobulin G1 Fc, are expressed with an N-terminal GFP fusion, allowing for structural studies using mass spectrometry and nuclear magnetic resonance spectroscopy.
The system utilizes a serum-free medium adaptable for expressing isotopically enriched proteins and carbohydrates. Additionally, the N-glycan composition can be modified by adding specific molecules without affecting yield.
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