American Cyanamid Company

Sotirios K Karathanasis

Paul Wai Mak

Elwood Eugene Largis

Florence Mahlberg

Sohail Malik

Andrew S Katocs, Jr

A method is described for screening retinoid X receptor agonists or antagonists using a retinoid X receptor expressed by a yeast expression system to screen a compound having a retinoid X receptor agonist or antagonist activity. Another screen is described for detecting a compound having retinoid X receptor agonist or antagonist activity by: (1) providing a yeast strain which expresses the retinoic acid receptor and activates a reporter plasmid containing apolipoprotein AI gene site A or a mutated variant thereof; (2) incubating the compound in suitable media and a colorless chromogenic substrate; and (3) examining the media for development of color. Mutated variants of apolipoprotein AI gene site A which respond selectively to the retinoid X receptor or respond to receptors other than RXR.alpha. are also described.

Mechanism-based screen for retinoid X receptor agonists and antagonists

Mechanism based screen for retinoid X receptor agonists and antagonists

The present invention describes a novel method for screening retinoid X receptor agonists or antagonists, comprising: using a retinoid X receptor expressed by a yeast expression system to screen a compound having a retinoid X receptor agonist or antagonist activity as well as a screen for detecting a compound having retinoid X receptor agonist or antagonist activity, which comprises the steps of (1) providing a yeast strain which expresses the retinoic acid receptor and activates a reporter plasmid containing apolipoprotein AI gene site A or a mutated variant thereof; (2) incubating the compound in suitable media and a colorless chromogenic substrate; and (3) examining the media for development of color.

Recombinant nucleic acid containing a response element

Apolipoprotein A1 is prepared in a T7 RNA polymerase/promoter bacterial expression system that optimizes yield and facilitates subsequent purification of the protein. Apolipoprotein A1 cDNA is cloned into an Escherichia coli plasmid containing a bacteriophage T7 RNA polymerase promoter such as a pT or pET plasmid; the recombinant plasmid is transferred to an E. coli strain containing inducible bacteriophage T7 RNA polymerase, e.g., a strain having the enzyme under lac control; T7 RNA polymerase is induced in the strain; and apolipoprotein A1 is isolated from the culture. Preferred embodiments tag the apolipoprotein with a sequence such as a stretch of histidines that facilitates purification subsequent to expression. An especially preferred embodiment employs a pET11d-6His plasmid and E. coli strain BL21(DE)pLysS that produces at least 1 mg protein per liter of culture, preferably up to 2 mg per liter, and the protein can be purified to homogeneity by one-step affinity chromatography on a nickel chelating resin.

High yield production of human apolipoprotein A1 in E. coli.

A method to increase plasma high density lipoprotein levels for the treatment and prevention of coronary artery disease by administering a therapeutic amount of a retinoid of the general formula: ##STR1##

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Rockland, NY, United States of America

Sotirios K Karathanasis