Journal of Pharmacological and Toxicological Methods
Appraisal of state-of-the-artHEK293 cell line: A vehicle for the expression of recombinant proteins
Section snippets
History
The transformation of human embryonic kidney (HEK) cells following exposure to sheared fragments of human adenovirus type 5 (Ad5) DNA generated the widely used expression tool known today as the HEK293 cell line (hereafter referred to as the HEK cell). This permanently transformed cell line has incorporated Ad5 into chromosome 19 of the host genome, a modification used subsequently for the generation of recombinant E1-deleted (i.e. transcription incompetent) human adenoviral vectors. Until
Methods for preparation and maintenance of HEK cells
HEK cells can be sourced from American Type Culture Collection (ATCC: Manassas, USA) under the designation ‘293’, accession code CRL-1573. As well as a repository for the original Ad5 transformed cell line, ATCC also maintain another variant called HEK 293E, which has been transformed by the Epstein–Barr (EBNA1) virus. This, along with an SV40 virus transformation product called HEK 293T, is not as widely used in expression studies as ‘293’. Purified genomic DNA from the ‘293’ cell line is also
Choice of cell
With a resting membrane potential (Rp) of around −40 mV the HEK cell clearly displays electrotonic properties which are somewhat different from most neurons. We would routinely voltage clamp a HEK cell at or near its Rp (i.e. generally −20 to −40 mV). Given that the chloride concentrations in our intra- and extracellular solutions are balanced (i.e. 0 mV equilibrium potential) across the membrane in which our GABA-gated chloride channel is expressed, a holding potential of −40 mV provides more
Advantages
The fidelity with which HEK cells are able to express exogenous receptor makes these cells amenable to many kinds of transfection procedure, permitting the expression of proteins from many sources and for many purposes. For instance, HEK cells have been used to express a wide variety of proteins important to the neuropharmacologist: voltage-sensitive ion channels (sodium, potassium, calcium), G-protein coupled receptors, ligand-gated ion channels, post-synaptic density proteins (PSD95,
Discussion
Overall, the HEK293 cell provides a robust and reliable platform in which to express receptor proteins and ion channels with high fidelity. These protein production rates are important if all or part of a protein is to be crystallised, hybrid screened for associated molecules, or used in biochemical assays. Though many of these scale-up procedures can be undertaken in yeast cells, because the HEK cell is amenable to rapid amplification of protein product, it too can be used to generate tens of
Acknowledgements
We are grateful to Martin Mortensen (UCL) for providing unpublished data for Fig. 3A and B, to Chris Connelly (University of Dundee) for providing scanning electron micrograph images for Fig. 1, A3, and to the MRC and The Wellcome Trust for support.
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