Methods comprising apoptosis inhibitors for the generation of transgenic pigs

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Stem Cell Related Patent Number US5641663

Title:Expression system for the secretion of bioactive human granulocyte macrophage colony stimulating factor (GM-CSF) and other heterologous proteins from steptomyces
Inventors:Garvin, Robert T.; Toronto, Canada
Summary:This invention describes a gene expression system comprised of a regulatory polynucleotide molecule that is operatively linked to a second polynucleotide molecule encoding a eucaryotic protein. Described herein are the genes for protease A and protease B from Streptomyces griseus and methods of their use in the expression of heterologous bioactive, glycosylated, oxidized fusion proteins such as the granulocyte macrophage colony stimulating factor (GM-CSF) which is secreted from a host selected from the Streptomyces genera. Further disclosed is a regulatory nucleotide sequence linked to a second nucleotide sequence encoding the heterologous protein, such that the regulatory sequence encodes a peptide which directs the secretion of the heterologous protein in bioactive form wherein the regulatory sequence includes a signal sequence and a promoter sequence. Also provided is a second nucleotide sequence encoding GM-CSF or a biologically active derivative thereof, which may be either natural or synthetic. Claims of the invention include similar constructs and novel signal peptides for the expression of interleukin 3 (IL-3), interleukin 6 (IL-6), tumor necrosis factor alpha (TNF?a), human stem cell factor (SCF), interleukin 7 (IL-7), erythropoietin (EPO) and interleukin 2 (IL-2).
Abstract:A gene expression system is used to produce heterologous biologically active proteins, in particular bioactive granulocyte macrophage colony stimulating factor (GM-CSF), secreted from a host selected from the Streptomyces genera. The gene expression system includes a regulatory nucleotide sequence linked to a second nucleotide sequence encoding the heterologous protein. The regulatory sequence, encodes a peptide which directs the secretion of the heterologous protein in bioactive form from a host selected from the Streptomyces genera. The regulatory sequence includes a signal sequence and a promoter sequence. The second nucleotide sequence, which encodes GM-CSF or a biologically active derivative of GM-CSF, may be either natural or synthetic. In particular, the invention relates to an expression system for secreting bioactive, non-glycosylated, oxidized, therapeutically useful GM-CSF from a host selected from the Streptomyces genera. Also disclosed are similar constructs for expression of interleukin 3 (IL-3), interleukin 6 (IL-6), tumor necrosis factor alpha (TNF.alpha.), human stem cell factor (SCF), interleukin 7 (IL-7), erythropoietin (EPO) and interleukin 2 (IL-2). Novel signal peptides of these constructs include hybrids of the signal peptides of Streptomyces griseus protease B and Escherichia coli omp A, hybrids of Streptomyces griseus protease B and Streptomyces limosus .alpha.-amylase signal peptide.
US Patent Website:Click Here for Full Text of Patent
Title Number:US5641663
Application Number:US1994000318193
Date Filed:05/10/1994
Date Published:24/06/1997
Assignee:Cangene Corporation, Mississauga, Canada


 
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