No abstract

Secreted placental alkaline phosphatase (SPAP) is commonly used as a reporter gene for the study of factors that control transcription. Expression of reporter constructs such as SPAP in mammalian cell lines give rise to reporter cell lines and these are routinely used by pharmaceutical companies for the discovery of new agents and to investigate by which these cells communicate with each other.Normally, secreted alkaline phosphatase is detected colourimetrically by monitoring the production of the yellow chromophore p-nitro phenol which is generated by the action of SPAP on the colourless substrate p-nitro phenol phosphate. In order to obtain more sensitive measurements, luminescent assay methods have recently been developed.An alternative approach to achieving the sensitivity of luminescent assays, hut at a fraction of the cost, is to use electrochemical detection strategies. A further advantage of this approach is that electrochemical systems lend themselves very well to recent developments in nanotechnology apd chip technology especially in terms of designing multiple sensor arrays on a single chip. This has implications in the important area of high throughput screening.This poster communicates preliminary data indicating that SPAP can indeed be measured electrochemically with good sensitivity at a mass produced, disposable carbon electrode system. Our goal is to produce and purify large amounts of biologically active human a2-C2 (a2B) adrenergic receptor for crystallisation experiments. To that end, we have pursued several different approaches. Thus far, the most advanced strategy is based on heterologous production in Saccharomyces crrrvrsiue followed hy detergent solubilisation, immunoaffinity purification and reconstitution to phospholipid vesicles. This way, we can produce a2-C2 adrenergic receptor in milligram amounts and purify it to apparent homogeneity, however, currently only in sub-milligram batches with modest total yield. Of total receptor protein, about 55 7% can he recovered after immunopurification and less than 10 7% of the purified protein can be reconstituted into phospholipid vesicles as active receptors.To increase the yield, we have pursued ways to improve all key points in the process: production levels, solubilisation efficiency and reconstitution yield. For higher production levels, improved yeast-based production systems have been developed, in addition to a Halobacterium -and virus-based mammalian cell expression system. In yeast systems, we are decreasing batch-to-batch variation in production level by using genetically more stable vector-host systems and stringently controlled promoters. In addition we aim to increased cell mass production in fermentation. The two other, very different systems, Halobacterium hahbium and Semliki Forest Virus/Baby Hamster Kidney cell system, represent the two extremes in the host range, offering different advantages and disadvantages. The archaean prokaryote is well known for its ability to produce enormous amounts of a 7-TM protein bacteriorhodopsi...