Campus Units

Biochemistry, Biophysics and Molecular Biology, Roy J. Carver Department of

Document Type

Article

Publication Version

Published Version

Publication Date

12-2015

Journal or Book Title

Journal of Visualized Experiments

Issue

106

First Page

e53568

DOI

10.3791/53568

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.

Comments

This article is published as Subedi, G.P., Johnson, R.W., Moniz, H.A., Moremen, K.W., Barb, A. High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension. J. Vis. Exp. (106), e53568, doi: 10.3791/53568 (2015). Posted with permission.

Copyright Owner

Journal of Visualized Experiments

Language

en

File Format

application/pdf

Share

COinS