The cDNAs encoding human ␦ (hDOR), (hKOR) and (hMOR) opioid receptors were cloned in the baculovirus Autographa californica (AcMNPV) under the control of the polyhedrin promoter with or without an amino-terminal hexahistidine tag. Expression levels were optimized in Spodoptera frugiperda (Sf9) cells and were in the following order hMOR > hDOR > hKOR. The receptors bound antagonists with affinity values similar to those published previously for the receptors expressed in mammalian cells. They also retained selectivity toward specific antagonists. The three receptors bound peptidic agonists with low affinity, suggesting that they might not be functionally coupled to intracellular effectors. Introduction of an amino-terminal hexahistidine tag decreased the levels of expression markedly. Only hMOR-his was expressed at a level allowing binding study, but no difference could be detected in the affinities of both agonists and antagonists compared with the nontagged protein. hMOR expression was also optimized in High Five cells leading to a further increase in protein production. The pharmacological profile was similar to the one obtained when the receptor was expressed in Sf9 cells. Our results show that the baculovirus expression system is suitable for large scale production of human opioid receptors.Opioid receptors and endogenous opioid peptides form a neuromodulatory system that plays a major role in the control of nociceptive pathways. The opioid system is not only a key element for pain perception but also modulates affective behavior as well as neuroendocrine physiology and controls autonomic functions such as respiration, blood pressure, thermoregulation, and gastrointestinal motility. It affects locomotor activity and could be involved in learning and memory. The receptors are otherwise targets for exogenous narcotic drugs, a major class of drugs of abuse.Genes coding for ␦, , and opioid receptor subtypes have now been identified and isolated from different vertebrates. Primary sequence analysis indicated that opioid receptors belong to the G-coupled receptor family whose structure shows a seven-transmembrane domain topology (for review, see Refs. 1 and 2). Engineering of protein chimeras together with generation of point mutants led to a better identification of the receptor regions interacting specifically with different ligands and/or involved in the signal transduction pathway. However, almost no structural data are available but only a few models based on rhodopsin and bacteriorhodopsin structures (2-5). Recently a new insight was brought by a model drawn without template for the ␦ receptor (6).The baculovirus expression system is extremely efficient to produce large amounts of mammalian proteins. Post-translational modifications are identical to those observed in mammalian cells with the exception of glycosylation, which is mostly of the high mannose type. Several G-coupled receptors, some of human origin, have already been expressed successfully under a functional state including adrenergic; ␥-aminobuty...

We produced bovine trypsinogen in the yeast Pichia pastoris. Little or no trypsinogen was detected when the gene with its native leader sequence was expressed under the control of the strong aox1 promoter, suggesting that expression of the wild-type bovine trypsinogen was toxic to the cells. We altered the trypsinogen native propeptide sequence by replacing the lysine at position 6 with an aspartic acid, thus destroying the site in the propeptide cleaved by enterokinase and by trypsin. This mutant accumulated up to 10 mg of trypsinogen per liter in shake flask cultures and about 40 mg/liter in 6-liter fermentors. Trypsinogen could be activated in vitro with a dipeptidyl-aminopeptidase, which selectively removed the modified trypsinogen propeptide; the resulting trypsin was fully active and showed evidence of glycosylation. Thus, we have developed a novel protein production scheme that can be used for the expression of proteins, such as proteases, that are deleterious to the producing organism. This system relies on the expression of a zymogen that cannot be activated in vivo coupled with its in vitro purification and activation.

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...