Large-scale functional expression of WT and truncated human adenosine A(2A) receptor in Pichia pastoris bioreactor cultures

Background: The large-scale production of G-protein coupled receptors (GPCRs) for functional and structural studies remains a challenge. Recent successes have been made in the expression of a range of GPCRs using Pichia pastoris as an expression host. P. pastoris has a number of advantages over other expression systems including ability to post-translationally modify expressed proteins, relative low cost for production and ability to grow to very high cell densities. Several previous studies have described the expression of GPCRs in P. pastoris using shaker flasks, which allow culturing of small volumes (500 ml) with moderate cell densities (OD600 similar to 15). The use of bioreactors, which allow straightforward culturing of large volumes, together with optimal control of growth parameters including pH and dissolved oxygen to maximise cell densities and expression of the target receptors, are an attractive alternative. The aim of this study was to compare the levels of expression of the human Adenosine 2A receptor (A(2A)R) in P. pastoris under control of a methanol-inducible promoter in both flask and bioreactor cultures. Results: Bioreactor cultures yielded an approximately five times increase in cell density (OD600 similar to 75) compared to flask cultures prior to induction and a doubling in functional expression level per mg of membrane protein, representing a significant optimisation. Furthermore, analysis of a C-terminally truncated A2AR, terminating at residue V334 yielded the highest levels (200 pmol/mg) so far reported for expression of this receptor in P. pastoris. This truncated form of the receptor was also revealed to be resistant to C-terminal degradation in contrast to the WT A(2A)R, and therefore more suitable for further functional and structural studies. Conclusion: Large-scale expression of the A(2A)R in P. pastoris bioreactor cultures results in significant increases in functional expression compared to traditional flask cultures.

Ischaemia differentially regulates GABAB receptor subunits in organotypic hippocampal slice cultures

Reduced synaptic inhibition due to dysfunction of ionotropic GABAA receptors has been proposed as one factor in cerebral ischaemia-induced excitotoxic cell death. However, the participation of the inhibitory metabotropic GABAB receptors in these pathological processes has not been extensively investigated. We used oxygen–glucose deprivation (OGD) and NMDA-induced excitotoxicity as models to investigate whether ischaemia-like challenges alter the protein levels of GABAB1 and GABAB2 receptor subunits in rat organotypic hippocampal slice cultures. Twenty-four hours after the insult both OGD and NMDA produced a marked decrease in the total levels of GABAB2 (75%), while there was no significant change in the levels of GABAB1 after OGD, but an increase after NMDA treatment (100%). The GABAB receptor agonist baclofen (100 μM) was neuroprotective following OGD or NMDA treatment if added before or during the insult. GABAB receptors comprise heterodimers of GABAB1 and GABAB2 subunits and our results suggest that the separate subunits are independently regulated in response to extreme neuronal stress. However, because GABAB2 is required for functional surface expression, down-regulation of this subunit removes an important inhibitory feedback mechanism under pathological conditions.

Doubled haploid ramets via embryogenesis of haploid tissue cultures

Tissue culture in the oil palm business is generally concerned with the multiplication (clonal production) of dura, pisifera and tenera palms. These are all normal diploids (2n=2x=36). Sumatra Bioscience has pioneered haploid tissue culture of oil palm (n=x=18). Haploid oil palm is the first step in producing doubled haploid palms which in turn provide parental lines for F1 hybrid production. Chromosome doubling is known to occur during embryogenesis in other haploid cultures, e.g. barley anther culture. Haploid tissue cultures in oil palm were therefore set up to investigate and exploit spontaneous chromosome doubling during embryogenesis. Flow cytometry of embryogenic tissue showed the presence of both haploid (n) and doubled haploid (2n) cells indicating spontaneous doubling. Completely doubled haploid ramets were regenerated suggesting that doubling occurred during the first mitoses of embryogenesis. This is the first report of doubled haploid production in oil palm via haploid tissue culture. The method provides a means of producing a range of doubled haploids in oil palm from the 1,000 plus haploids available at Sumatra Bioscience, in addition the method also produced doubled haploid (and haploid) clones. 1.