Recombinant expression of Aryl Hydrocarbon Receptor for quantitative ligand-binding analysis

Recombinant expression of the Aryl Hydrocarbon Receptor (AhR) yields small amounts of ligand- binding competent AhR. Therefore, Spodoptera frugiperda (Sf9) cells and baculovirus have been evaluated for high level and functional expression of AhR. Rat and human AhR were expressed as soluble protein in significant amounts. Expression of ligand-binding competent AhR was sensitive to the protein concentration of Sf9 extract, and co-expression of the chaperone p23 failed to affect the yield of functional ligand-binding AhR. The expression system yielded high levels of functional protein, with the ligand-binding capacity (Bmax) typically 20- fold higher than that obtained with rat liver cytosol. Quantitative estimates of the ligand-binding affinity of human and rat AhR were obtained; the Kd for recombinant rat AhR was indistinguishable from that of native rat AhR, thereby validating the expression system as a faithful model for native AhR. The human AhR bound TCDD with significantly lower affinity than the rat AhR. These findings demonstrate high-level expression of ligand-binding competent AhR, and sufficient AhR for quantitative analysis of ligand-binding.

DNA charge neutralisation by linear polymers II: reversible binding

We model the way in which polymers bind to DNA and neutralise its charged backbone by analysing the dynamics of the distribution of gaps along the DNA. We generalise existing theory for irreversible binding to construct new deterministic models which include polymer removal, movement along the DNA and allow for binding with overlaps. We show that reversible binding alters the capacity of the DNA for polymers by allowing the rearrangement of polymer positions over a longer timescale than when binding is irreversible. When the polymers do not overlap, allowing reversible binding increases the number of polymers adhered and hence the charge that the DNA can accommodate; in contrast, when overlaps occur, reversible binding reduces the amount of charge neutralised by the polymers.