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• W2493209358 abstract "Cyclic nucleotides (cAMP and cGMP) are second messenger molecules that play a key role in many physiological processes, cAMP in particular regulates cell growth, cell differentiation, inflammation and glycogen metabolism. cAMP is degraded by phosphodiesterase 3 (PDE3) and phosphodiesterase 4 (PDE4) which belong to a large family of PDEs consisting of 12 members. PDE4 isoenzymes which are highly specific for cAMP are most abundant in inflammatory cells making them good clinical targets for the therapy of asthma and other inflammatory disorders. The design of potential therapeutics will be helped by the structural analysis of PDE4. To date, there have been limited reports on expression systems capable of producing milligram quantities of full length PDE4. This has been due to the protein's paucity and susceptibility to proteolysis. Although, the catalytic domain of PDE4 has been recently analysed by X-ray crystallography (Xu et al, 2000), information regarding the regulation of the enzyme by its N-terminal regulatory domain and the binding of rolipram a PDE4 specific inhibitor is still poorly understood. Therefore, the main objective of this work was to establish a suitable expression system capable of expressing full length cAMP-specific phosphodiesterase 4 in quantities necessary to crystallise and analyze by X-ray crystallography. To achieve this objective Met26RD1 clone was used. This clone consisted of rat brain cAMP-specific phosphodiesterase 4 gene which was cloned originally using Drosophila dunce gene as a probe hence the name RD1 (rat dunce 1). The PDE4 gene consisted of both regulatory and catalytic domains but was missing the first 25 amino acids hence the name Met26 which helped solubilisation and prevented membrane attachment. PDE gene Met26RD1 was cloned and expressed in two systems, bacterial and mammalian. The bacterial system expressed Met26RD1 as a Glutathione S-transferase fusion protein in Escherichia coli. A substantial portion of phosphodiesterase expressed in bacteria was found to be inactive, insoluble and very highly degraded by proteolytic enzymes. Consequently, PDE4 expression in other systems was investigated. However, the insoluble protein produced was solubilised in urea and used to raise antibodies in rabbits as there were no commercial antibodies available against this protein. Phosphodiesterase was also expressed in mammalian cells using Semliki Forest Virus (SFV) system. SFV system parameters were optimised for BHK-21 cells using β-galactosidase gene due to the ease in which cells expressing this gene could be assayed and analysed in-situ compared to cells expressing the Met26RD1 gene. Semliki Forest viral particles carrying Met26RD1 mRNA were produced by co-transfection of BHK-21 cells with pSFV-1 Met 26RD1 mRNA and packaging-deficient helper RNA molecules at a ratio of 1; 1. Met26RD1 was expressed by infecting BHK-21 cells with the recombinant virus at a multiplicity of infection of 0.5-1. This produced approximately 0.1-0.3 mg of Met26RD1 protein per 107 cells (75-90 ml) with Km of 7.9 ± 0.86 μM and IC50 of 0.7 μM to rolipram (PDE4 selective inhibitor) consistent with previously published data for Met26RD1 (Shakur et al., 1993). Preliminary purification strategy to purify Met26RD1 protein was developed using immunoaffinity column prepared from the antibody produced against the bacterial expressed Met26RD1 protein." @default.
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• W2493209358 date "2003-01-01" @default.
• W2493209358 modified "2023-09-28" @default.
• W2493209358 title "Expression studies of recombinant cAMP-specific phosphodiesterase type 4" @default.
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