High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension

Journal of Visualized Experiments : JoVE
Ganesh P SubediAdam Barb

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 mo...Continue Reading

Citations

Nov 1, 2017·International Journal of Molecular Sciences·Ying XuShuqing Chen
Aug 9, 2018·Scientific Reports·David F ThiekerRobert J Woods
Jan 25, 2020·Molecular Medicine Reports·Qi ChenQing Yuan
Aug 10, 2019·Nature Communications·Ren-You PanShuen-Iu Hung
Sep 2, 2020·Wellcome Open Research·Andrea LiaPietro Roversi
Jul 1, 2017·PloS One·Melinda S HanesRichard D Cummings
Feb 18, 2020·Bioelectrochemistry·Nina KleinAntoni Ivorra
Nov 5, 2020·International Journal of Molecular Sciences·Masato OokaMenghang Xia
Sep 11, 2019·ACS Synthetic Biology·Hao PanMatthew J Paszek

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