title: High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension.
abstract: The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and function. The rapid establishment and high recovery from transiently-transfected mammalian cell lines addresses this barrier and is an effective means of expressing proteins that are naturally channeled through the ER and Golgi-mediated secretory pathway. Here is one protocol for protein expression using the human HEK293F and HEK293S cell lines transfected with a mammalian expression vector designed for high protein yields. The applicability of this system is demonstrated using three representative glycoproteins that expressed with yields between 95-120 mg of purified protein recovered per liter of culture. These proteins are the human FcγRIIIa and the rat α2-6 sialyltransferase, ST6GalI, both expressed with an N-terminal GFP fusion, as well as the unmodified human immunoglobulin G1 Fc. This robust system utilizes a serum-free medium that is adaptable for expression of isotopically enriched proteins and carbohydrates for structural studies using mass spectrometry and nuclear magnetic resonance spectroscopy. Furthermore, the composition of the N-glycan can be tuned by adding a small molecule to prevent certain glycan modifications in a manner that does not reduce yield.
authors.affiliation: The Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University.
authors.forename: Ganesh P
authors.initials: GP
authors.lastname: Subedi
authors.affiliation: Complex Carbohydrate Research Center, University of Georgia.
authors.forename: Roy W
authors.initials: RW
authors.lastname: Johnson
authors.affiliation: Complex Carbohydrate Research Center, University of Georgia.
authors.forename: Heather A
authors.initials: HA
authors.lastname: Moniz
authors.affiliation: Complex Carbohydrate Research Center, University of Georgia.
authors.forename: Kelley W
authors.initials: KW
authors.lastname: Moremen
authors.affiliation: The Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University; abarb@iastate.edu.
authors.forename: Adam W
authors.initials: AW
authors.lastname: Barb
mesh_terms: D000818:Animals; D002846:Chromatography, Affinity; D006023:Glycoproteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D057809:HEK293 Cells; D006801:Humans; D011499:Protein Processing, Post-Translational; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D014162:Transfection / Q000379:methods*
chemical_list: D006023:Glycoproteins; D011994:Recombinant Proteins