In the course of our ongoing attempts to

In the course of our ongoing attempts to photolabel allosteric binding sites of γ-aminobutyric estrone type A (GABAA) receptors, we were met with the need for relatively large (near mg) quantities of recombinant protein. Rather than establishing a stable cell line we sought to optimise the process of transient transfection of HEK293 cells, with the intent of permitting rapid expression of a variety of receptor isoforms. As the majority of existing studies were tailored for the pharmaceutical production of antibodies and other therapeutic proteins, a reappraisal of techniques was necessary to determine the ideal conditions for transfection and expression of IMPs. We employed [3H]muscimol binding as a direct means of determining GABAA receptor expression, with multiple variables assessed including the cell line, vector, medium formulation, harvest time, and transfection protocol. Following four rounds of optimisation an approximate 40 fold improvement in productivity was achieved. Although one previous study has reported the optimisation of transfection method of GABA receptors in HEK cells [34], to our knowledge this is the first systematic evaluation of multiple factors affecting the transient expression of IMPs in mammalian cells.
Materials and methods
Results and discussion
Conclusions While transient transfection platforms have evolved to become a viable alternative to stable cell lines for the production of secreted proteins [55,76], they are evidently not as amenable to the overexpression of membrane proteins. Through the application of a nanoparticle transfection protocol, techniques employed in the biopharmaceutical sector, and design of a novel pARVUS/pARVA vector system, we were able to achieve a 40 fold improvement in the transient expression of GABAA receptors in HEK293 cells. While this method was developed during the course of our ongoing attempts to photolabel allosteric binding sites, we anticipate that the rapidly interchangeable nature of this technique will assist in future structural studies of a variety of IMPs. On a final note, it should be considered that although we successfully narrowed down the optimum conditions for transient transfection of GABAA receptors, employing a “one-factor-at-a-time” approach severely limits the number of variables which may be assessed. Measuring cell viability and the equal loading of protein for each sample may have provided insight into the effects of transfection conditions on cell health versus productivity. Additionally, assessing the percentage of aggregated receptors by detergent solubilisation may have uncovered effects on protein folding. There also remains the pertinent question of what acts as the major bottleneck to IMP overexpression, and whether this differs from soluble intracellular and secreted proteins. While these points were beyond the scope of the current study, we hope that they will be addressed by further work in the field.
Funding The authors acknowledge funding from the National Health and Medical Research Council.
Acknowledgements S.O.D. acknowledges the support of an Australian Postgraduate Award and John A. Lamberton Scholarship.
Introduction The hippocampus is a key region associated with the pathophysiology of epilepsy (Jutila et al., 2002). There are two distinct categories of GABA-A receptors in the hippocampus. Synaptic receptors and extrasynaptic receptors exhibit different characteristics in their affinity and efficacy to GABA, desensitization rate, and drug sensitivity (Bianchi and Macdonald, 2002, Bianchi and Macdonald, 2003, Brown et al., 2002, Wohlfarth et al., 2002, Mortensen et al., 2011). Synaptic receptors, mainly γ-containing receptors, produce rapid and transient phasic currents in response to presynaptic release of GABA (∼1 mM), whereas extrasynaptic receptors, mostly δ-containing receptors in dentate gyrus and α5β3γ2 receptors in CA1, generate persistent, non-desensitizing tonic currents by ambient GABA (~ 0.3 – 2 μM) (Mody et al., 1994, Brickley et al., 1996, Farrant and Nusser, 2005). Tonic currents contribute to the overall basal tone and shunting inhibition via continuous channel conductance in neurons expressing δ-containing receptors. Hippocampus dentate gyrus granule cells (DGGCs), which express high density of δ-subunit-containing extrasynaptic receptors, promote tonic inhibition and thereby regulate network excitability (Coulter and Carlson, 2007, Glykys et al., 2008). GABA-A receptors are modulated by many compounds including benzodiazepines, neurosteroids, and zinc (Zn2+).