cholecystokinin and Respiration-Disorders

Membrane proteins are targets of most available pharmaceuticals, but they are difficult to produce recombinantly, like many other aggregation-prone proteins. Spiders can produce silk proteins at huge concentrations by sequestering their aggregation-prone regions in micellar structures, where the very soluble N-terminal domain (NT) forms the shell. We hypothesize that fusion to NT could similarly solubilize non-spidroin proteins, and design a charge-reversed mutant (NT*) that is pH insensitive, stabilized and hypersoluble compared to wild-type NT. NT*-transmembrane protein fusions yield up to eight times more of soluble protein in Escherichia coli than fusions with several conventional tags. NT* enables transmembrane peptide purification to homogeneity without chromatography and manufacture of low-cost synthetic lung surfactant that works in an animal model of respiratory disease. NT* also allows efficient expression and purification of non-transmembrane proteins, which are otherwise refractory to recombinant production, and offers a new tool for reluctant proteins in general.

Topics: Animals; Cholecystokinin; Chromatography; Circular Dichroism; Dimerization; Disease Models, Animal; Escherichia coli; Female; Fibroins; Hydrogen-Ion Concentration; Lung; Magnetic Resonance Spectroscopy; Micelles; Microscopy, Electron, Transmission; Mutagenesis, Site-Directed; Mutation; Peptides; Protein Domains; Rabbits; Recombinant Proteins; Respiration Disorders; Silk; Spiders; Surface-Active Agents

The effects of the cholecystokinin antagonist devazepide on analgesia and respiratory depression induced by morphine in squirrel monkeys were examined. Pain thresholds were determined using the tail withdrawal procedure, in which monkeys restrained in chairs kept their tails in cool (35 degrees C) water for at least 20 sec, but withdrew them from warm (55 degrees C) water in less than 4 sec. Morphine produced a dose-related increase in tail withdrawal latencies from warm water. Devazepide (injected i.p. or p.o.) had no effect on tail withdrawal latencies when given alone but enhanced the analgesic effects of morphine. The devazepide dose-response curve for morphine enhancement was bell-shaped with doses of 3, 10, 30 and 100 micrograms/kg injected i.p. increasing morphine analgesia whereas higher and lower dose did not. In a separate group of monkeys, morphine produced dose-dependent decreases in respiratory rate and oxygen tension and increases in carbon dioxide tension. In contrast to its effects on morphine analgesia, devazepide had no effect on the various indices of morphine-induced respiratory depression. These data suggest that devazepide may have therapeutic utility as an adjuvant to morphine analgesia allowing lower dose of the opiate to be used to relieve pain and reducing the risk of opiate-induced respiratory depression.

Topics: Administration, Oral; Analgesia; Animals; Benzodiazepinones; Cholecystokinin; Devazepide; Dose-Response Relationship, Drug; Drug Synergism; Injections, Intraperitoneal; Male; Morphine; Naloxone; Pain; Pain Measurement; Receptors, Cholecystokinin; Respiration Disorders; Saimiri