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) 293F cells.
Highlights
The rapid progress of molecular cell biology and the constant need for improved drugs in medicine has created a need for structural biologists to look at increasingly more complex protein structures
Histone Deacetylase 1 (HDAC1) and Suppressor of Defective Silencing 3 (SDS3) interact with Sin3A through the HDAC-interaction domain (HID) of Sin3A
We have developed a straightforward and cost effective method (PEI costs much less than commercially available lipophilic transfection reagents) for expressing and purifying large amounts of recombinant proteins and multi-subunit complexes from mammalian cells
Summary
The rapid progress of molecular cell biology and the constant need for improved drugs in medicine has created a need for structural biologists to look at increasingly more complex protein structures. Cells are transiently transfected using a branched version of polyethylenimine (PEI), an inexpensive polymeric reagent that has been reported to function for a large range of mammalian cells[4] by forming DNA/PEI complexes that enter the host cell by endocytosis[5] This method is suitable for both small scale (30 ml) and large-scale (up to 300 ml) experiments and can produce high levels of purified protein complexes. It is useful for studying proteins that require complex folding machineries, co-factors or particular post-translational modifications that cannot be performed by bacteria, yeast and insect cells. The atmosphere will need to be replaced at each passage (normally every 2 days)
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