The Salk Institute for Biological Studies

Steven T Suhr

Fred Harrison Gage

Elad B Gil

Marie-Claude C Senut

The invention provides chimeric proteins having at least two functional protein units, each containing the dimerization domain of a member of the steroid/thyroid hormone nuclear receptor superfamily. The chimeric proteins can fold under crystallization conditions to form functional entities. The functional entities optionally contain a novel flexible peptide linker of variable lengths between at least two of the protein units. In a preferred embodiment, the linker is designed to be increased in increments of 12 amino acids each to aid in preparation of variant chimeric proteins. The DNA binding characteristics of the invention functional entities differ from those of wild-type complexes formed between 'monomeric' receptors and their binding partners. Some functional entities, e.g. dimers expressed as fusion proteins, transactivate responsive promoters in a manner similar to wild-type complexes, while others do not promote transactivation and function instead essentially as constitutive repressors. The invention further provides nucleotide sequences encoding the invention chimeric proteins, cells containing such nucleotide sequences, and methods for using the invention chimeric proteins to modulate expression of one or more exogenous genes in a subject organism. In addition, isolated protein crystals suitable for x-ray diffraction analysis and methods for obtaining putative ligands for the invention chimeric proteins are provided.

Hormone receptor functional dimers and methods of their use

In accordance with the present invention, it has been discovered that nuclear receptor proteins isolated from the silk moth(bR) are useful for the regulation of transgene expression. bR is generally thought to be a strong transcriptional regulator within cells of the silk moth. In accordance with the present invention, it has been discovered that bR is also functional in mammalian cells. It has further been discovered that the addition of activation domains to the bR open-reading frame enhances the activity of the ligand modulated regulator to afford high-level transcriptional induction. Further modifications to the bR ligand binding domain result in receptors with unique transactivational characteristics.

Modified lepidopteran receptors and hybrid multifunctional proteins for use in transcription and regulation of transgene expression

In accordance with the present invention, it has been discovered that nuclear receptor proteins isolated from the silk moth,(bR) are useful for the regulation of transgene expression. bR is generally thought to be a strong transcriptional regulator within cells of the silk moth. In accordance with the present invention, it has been discovered that bR is also functional in mammalian cells. It has further been discovered that the addition of activation domains to the bR open-reading frame enhances the activity of the ligand modulated regulator to afford high-level transcriptional induction. Further modifications to the bR ligand binding domain result in receptors with unique tranactivational characteristics.

In accordance with the present invention, novel retroviral vectors containing modified long terminal repeats (LTRS) which enable high level and ligand-modulatable expression of a desired gene product, even after prolonged periods of cellular quiescence, have been designed and constructed. Invention vectors overcome proviral transcriptional inactivation which occurs in cultured primary cells that are growth arrested due to environmental constraints such as contact inhibition and/or nutrient starvation. Invention vectors represent a class of retroviral vectors in which LTR-promoted proviral expression in infected cells may be maintained or increased, even in situations generally considered to be non-permissive for retroviral vectors.

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La Jolla, CA, United States of America

Steven T Suhr