Appraisal of state-of-the-art
HEK293 cell line: A vehicle for the expression of recombinant proteins

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Abstract

The HEK cell line has been extensively used as an expression tool for recombinant proteins since it was generated over 25 years ago. Although of epithelial origin, its biochemical machinery is capable of carrying out most of the post-translational folding and processing required to generate functional, mature protein from a wide spectrum of both mammalian and non-mammalian nucleic acids. Though popular as a transient expression system, this cell type has also seen wide use in stably transfected forms (i.e. transformed cells) to study a variety of cell-biological questions in neurobiology. The principal attributes which have made the HEK cell a popular choice among electrophysiologists to study isolated receptor channels include; its quick and easy reproduction and maintenance; amenability to transfection using a wide variety of methods; high efficiency of transfection and protein production; faithful translation and processing of proteins; and small cell size with minimal processes appropriate for voltage-clamp experimentation. These, and other attributes, also mean that complementary biochemical/cell biological evaluations of expressed proteins can be performed in concert with functional analyses to establish detailed pharmacological and biophysical profiles for the action of new drugs and their targets. The increased amount of sequence information available from the human genome has placed greater emphasis upon heterologous cell expression systems as targets for high throughput structure–function evaluation of novel drug targets and disease markers. Here we have highlighted some of the innate characteristics of the HEK cell in order that its suitability as a vehicle for the expression of a gene product can be assessed for particular needs. We have also detailed some of the standard methods used for transfection and obtaining functional data from electrophysiological recording techniques.

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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