American Cyanamid Company

Basf Aktiengesellschaft

Wyeth

Bio-technology General Corporation

Deborah Tardy Chaleff

Donald R Kirsch

Bradley Alton Ozenberger

Mark Henry Pausch

John Richard Hadcock

Eileen Marie Kajkowski

Laura Price

Laura A Price

Mark Edward Ruppen

Susan Mancini Cady

Brian Lee Buckwalter

John S Logan

Gerald W Stockton

Meir Fischer

Chengjin Huang

Michael R Lebens

Jerome Stephens

The present invention relates to the complete biosynthetic pathway for the formation of the LL-F28249 compounds and, most importantly, the major component LL-F28249&#x3b1;. The purified and isolated nucleic acid molecule encoding the proteins of the biosynthetic pathway, which is isolated from a wild-type or mutant, is fully described in FIG.to FIG.-and SEQ ID NO:1. The DNA gene cluster and its expression in a suitable host enable the efficient production of the highly active natural metabolites and semisynthetic derivatives. The invention further concerns plasmids, vectors and host cells that contain and express the novel nucleic acid molecule. Of particular interest, the entire biosynthetic pathway fits compactly in three plasmids, Cos11, Cos36 and Cos40. The invention also concerns the purified and isolated biosynthesis proteins that are encoded by the whole DNA gene cluster. Additionally, the invention involves a new efficient, biochemical method of preparing moxidectin.

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Expression vectors and yeast cells that contain a heterologous G protein-coupled receptor gene and a gene mutation that causes increased sensitivity to receptor activation or a gene mutation that permits transcriptional activation of pheromone-responsive genes without cell cycle arrest. Methods of making the yeast cells.

Heterologous G protein coupled receptors expressed in yeast, their fusion with G proteins and use thereof in bioassay

A first aspect of the present invention is directed to a transformed yeast cell containing a first heterologous DNA sequence which codes for a G protein-coupled receptor, for example, the somatostatin receptor, and a second heterologous DNA sequence which codes for a G protein a subunit or portions thereof fused to DNA sequences from the yeast G protein in a subunit. A second aspect of the present invention is a transformed yeast cell containing a heterologous DNA sequence which codes for a G protein coupled receptor. A third aspect of the present invention is a method of assaying compounds to determine effects on cell growth.

The present invention is directed to expression vectors and yeast cells transformed therewith containing a first heterologous nucleotide sequence which codes for a G protein-coupled receptor, for example, the somatostatin receptor, and a second nucleotide sequence which codes for all or a portion of a G protein &agr;&bgr;&ggr; complex. Said heterologous protein is physically expressed in a host cell membrane in proper orientation for both stereoselective binding of ligands, as well as functional interaction with G proteins on a cytoplasmic side of the cell membrane. In some embodiments, a nucleotide sequence encoding a heterologous or chimeric G&agr; protein is expressed in conjunction with nucleotide sequences from the yeast G protein &bgr;&ggr; subunits. A second aspect of the present invention provides expression vectors and yeast cells transformed therewith encoding chimeric yeast/heterologous G protein coupled receptors. A third aspect of the present invention is directed to methods of assaying compounds using such expression constructs and yeast cell expression systems to determine the effects of ligand binding to the heterologous receptors expressed in the systems.

Method for detecting ligand binding to G protein coupled receptors

Novel modified or derivatized recombinant animal somatotropins. Methods for stabilizing recombinant animal somatotropins by modification or deletion of the Cysteine residues utilizing site directed mutagenesis to replace from one to four of the cysteine amino acid residues of said somatotropins with one or more different amino acid residues or by derivatization of (1) both cysteine amino acid residues in the small loop of said somatotropin, both cysteine amino acid residues in the large loop or the four cysteine amino acid residues in both loops of said somatotropin.

Stabilization of somatotropins by modification of cysteine residues

The present invention is directed to a expression vectors and yeast cells transformed therewith containing a first heterologous nucleotide sequence which codes for a G protein-coupled receptor, for example, the somatostatin receptor, and a second nucleotide sequence which codes for all or a portion of a G protein .alpha..beta..gamma. complex. Said heterologous protein is physically expressed in a host cell membrane in proper orientation for both stereoselective binding of ligands, as well as functional interaction with G proteins on the cytoplasmic side of the cell membrane. In some embodiments, a nucleotide sequence encoding a heterologous or chimeric G.alpha. protein is expressed in conjunction with nucleotide sequences from the yeast G protein .beta..gamma. subunits. A second aspect of the present invention provides expression vectors and transformed yeast cells encoding chimeric yeast/heterologous G protein coupled receptors. A third aspect of the present invention is directed to methods of assaying compounds using these expression constructs and yeast cell expression systems to determine the effects of ligand binding to the heterologous receptors expressed in the systems.

Recombinant expression vectors for expression of heterologous proteins

A first aspect of the present invention is directed to a transformed yeast cell containing a first heterologous DNA sequence which codes for a G protein-coupled receptor, for example, the somatostatin receptor, and a second heterologous DNA sequence which codes for a G protein .alpha. subunit or portions thereof fused to DNA sequences from the yeast G protein a subunit. A second aspect of the present invention is a transformed yeast cell containing a heterologous DNA sequence which codes for a G protein coupled receptor. A third aspect of the present invention is a method of assaying compounds to determine effects on cell growth.

Transformed yeast cells expressing heterologous G-protein coupled

The present invention relates to somatotropin analogues with amino acid changes in the alpha-helix 1 regions of said somatotropins, alone or in combination with mutations in the alpha-helix 3 and/or alpha-helix 2 regions, plus combinations with other changes to the native amino acid sequence of somatotropins. The resulting analogues are stable for formulation in sustained release formulations, while maintaining biological activity. Further, methods for conducting site-directed mutagenesis on DNA encoding proteins and/or polypeptide also are provided.

Somatotropins with alterations in the alpha-helix 1 region, and

The present invention relates to somatotropin analogues with amino acid changes in the .alpha.-helix 3 regions of said somatotropins, changes in the .alpha.-helix 2 regions, combinations thereof plus combinations with other changes to the native amino acid sequence of somatotropins. The resulting analogues are stable for formulation in sustained release, formulations, while maintaining biological activity. Further, methods for conducting site-directed mutagenesis on DNA encoding proteins and/or polypeptides also are provided.

Growing community of inventors

Pennington, NJ, United States of America

Deborah Tardy Chaleff