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|Title:||Recombinant protein expression for structural biology in HEK 293F suspension cells: a novel and accessible approach|
Watson, Peter J.
Millard, Christopher J.
Milano, Charles P..
Cowley, Shaun M.
Schwabe, John W. R.
|Citation:||Journal of Visualised Experiments, 2014 (92), pp. e51897|
|Abstract:||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.|
|Rights:||Copyright © 2014 JoVE. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-commercial License (http://creativecommons.org/licenses/by-nc/2.0/ ), which permits unrestricted use, distribution, and reproduction in any medium non-commercially, provided the original author and source are credited.|
|Appears in Collections:||Published Articles, Dept. of Biochemistry|
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