Recombinant protein expression for structural biology in HEK 293F suspension cells: a novel and accessible approach..pdf (656.91 kB)
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Recombinant protein expression for structural biology in HEK 293F suspension cells: a novel and accessible approach

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journal contribution
posted on 14.10.2015, 10:47 by Nicola Portolano, Peter J. Watson, Louise Fairall, Christopher J. Millard, Charles P.. Milano, Yun Song, Shaun M. Cowley, John W. R. Schwabe
The expression and purification of large amounts of recombinant protein complexes is an essential requirement for structural biology studies. For over two decades, prokaryotic expression systems such as E. coli have dominated the scientific literature over costly and less efficient eukaryotic cell lines. Despite the clear advantage in terms of yields and costs of expressing recombinant proteins in bacteria, the absence of specific co-factors, chaperones and post-translational modifications may cause loss of function, mis-folding and can disrupt protein-protein interactions of certain eukaryotic multi-subunit complexes, surface receptors and secreted proteins. The use of mammalian cell expression systems can address these drawbacks since they provide a eukaryotic expression environment. However, low protein yields and high costs of such methods have until recently limited their use for structural biology. Here we describe a simple and accessible method for expressing and purifying milligram quantities of protein by performing transient transfections of suspension grown HEK (Human Embryonic Kidney) 293 F cells.

History

Citation

Journal of Visualised Experiments, 2014 (92), pp. e51897

Author affiliation

/Organisation/COLLEGE OF MEDICINE, BIOLOGICAL SCIENCES AND PSYCHOLOGY/MBSP Non-Medical Departments/Molecular & Cell Biology

Version

VoR (Version of Record)

Published in

Journal of Visualised Experiments

eissn

1940-087X

Copyright date

2014

Available date

14/10/2015

Publisher version

http://www.jove.com/video/51897/recombinant-protein-expression-for-structural-biology-hek-293f

Language

en