Cangene Corporation

Akzo Nobel N.v.

Signalgene Inc.

Genescape Inc.

Lawrence T Malek

Cheryl Davey

Daniel Bartfeld

Robert T Garvin

Dany Hadary

Timothy J Krieger

Gisela Soostmeyer

David L Jenish

Michael J Butler

Roy Sooknanan

Eva Walczyk

Sheila Garven

Roy R Sooknanan

Phyllis Krygsman

Peter Franklin Lens

Eric James

Eva Walcyzk

Graham Henderson

Frits Wielaard

A kit is provided for preferentially amplifying target RNA in a sample of tester RNA relative to non-target RNA in the sample, the kit driver sequences complementary to the non-target tester RNA under conditions where the driver sequences hybridize to the non-target RNA, a nucleic acid primer capable of hybridizing to the target RNA under conditions suitable for extension of the nucleic acid primer; and a promoter template capable of hybridizing to a DNA that is complementary to the target RNA under conditions suitable for extension of the complementary DNA such that a functional double promoter is formed.

Subtractive amplification kit useful in the diagnosis of genetic disease mutation or variation

An aminopeptidase inhibitor is used when expressing heterologous protein in a bacterial host, such as Streptomyces. Use of such an inhibitor inhibits degradation of the heterologous protein by aminopeptidases. Inhibitors are designed based upon the mechanism and substrate specificity of the target protease and expressed protein.

Method for expression of proteins in bacterial host cells

This invention relates to a process for amplifying a specific nucleic acid sequence. The process involves synthesizing single-stranded RNA, single-stranded DNA and double-stranded DNA. The single-stranded RNA is a first template for a first primer, the single-stranded DNA is a second template for a second primer, and the double stranded DNA is a third template for synthesis of a plurality of copies of the first template. A sequence of the first primer or the second primer is complementary to a sequence of the specific nucleic acid and a sequence of the first primer or the second primer is homologous to a sequence of the specific nucleic acid. The amplification process may be used to increase the quantity of the specific nucleic acid sequence to allow detection, or to increase the purity of the specific nucleic acid sequence as a substitute for conventional cloning methodology.

Nucleic acid amplification process

A family of proteases endogenous to Streptomyces cells degrades heterologous proteins secreted from Streptomyces host cells. The previously unidentified proteases include (1) tripeptidyl aminopeptidase--Streptomyces ('Tap'), (2) a Streptomyces protease ('Ssp') which displayed significant amino acid sequence homology to Subtilisin BPN' and showed an ability to remove tripeptides from the amino termini of proteins and peptides, and (3) other proteases derived from Streptomyces which degraded certain substrates under certain conditions. Degradation was alleviated by selective inhibition of secreted proteases or by using hosts with impaired capabilities to produce proteases. An irreversible inhibitor was designed based upon the mechanism and substrate specificity of the target protease. Hosts secreting high amounts of proteases were selected. Impaired hosts were produced by deleting or altering the nucleotide sequence for the proteases.

Streptomyces proteases and methods for improved secretion of

A method is provided for preferentially amplifying target RNA relative to non-target RNA in a sample of tester RNA. According to the method, a sample of tester RNA is contacted with driver sequences which are complementary to the non-target RNA under conditions where the driver sequences hybridize to the non-target RNA. A nucleic acid primer is then extended using the target RNA as a template, forming a DNA template complementary to some or all of the target RNA. The DNA template formed using the target RNA as template is then rendered single-stranded to enable the hybridization of the DNA template to a promoter template. The DNA template is then extended using the promoter template to form an extended DNA template comprising a functional double-stranded promoter. Multiple copies of the target RNA sequence can now be transcribed from the extended DNA template through recognition of the contained functional double-stranded promoter by RNA polymerase.

Subtractive amplification

This invention relates to a process for amplifying a specific nucleic acid sequence or its complement at a relatively constant temperature and without serial addition of reagents. The process provides in a single reaction medium an RNA polymerase, DNA polymerase, a ribonuclease that hydrolyses RNA of an RNA-DNA hybrid without hydrolysing single or double-stranded RNA or DNA, and ribonucleoside and deoxyribonucleoside triphosphates. The process then provides an RNA first template in the reaction medium. The RNA first template comprises a sequence complementary to a specific nucleic acid sequence, minus-sense sequences for a promoter and initiation site that are recognized by the RNA polymerase, and a 5'-terminal sequence that is complementary to at least the minus-sense sequence of the initiation site. Thus, the RNA first template has an inverted repeat sequence which could fold into a 5'-terminal stem-loop structure. The DNA polymerase uses the RNA first template to synthesize a DNA second template that together comprise an RNA-DNA hybrid. The DNA second template has plus-sense sequences of the promoter and the initiation site, and a 3'-terminal priming sequence that is complementary to the plus-sense sequence of the initiation site. The ribonuclease then hydrolyses an RNA which comprises the RNA-DNA hybrid, allowing the 3'-terminal priming sequence to hybridize to the plus-sense sequence of the initiation site in the DNA second template. The DNA polymerase then uses the DNA second template to synthesize the promoter by extending the 3'-terminal priming sequence of the DNA second template. The resulting partially double-stranded DNA has a promoter oriented toward the apex of a stem-loop structure. The RNA polymerase then recognizes the promoter and transcribes the DNA second template, thereby providing copies of the RNA first template. The process thereafter maintains the reaction conditions for a time sufficient to achieve a desired amplification of the specific nucleic acid sequence or its complement. This invention includes a kit containing the reagents of this invention.

Terminal repeat amplification method

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.

Expression system for the secretion of bioactive human granulocyte

A family of proteases endogenous to Streptomyces cells degrade heterologous proteins secreted from Streptomyces host cells. The previously unidentified proteases include (1) tripeptidyl aminopeptidase--Streptomyces ('Tap'), (2) a Streptomyces protease ('Ssp') which displayed significant amino acid sequence homology to Subtilisin BPN' and showed an ability to remove tripeprides from the amino termini of proteins and peptides, and (3) other proteases derived from Streptomyces which degraded certain substrates under certain conditions. Degradation was alleviated by selective inhibition of secreted proteases or by using hosts with impaired capabilities to produce proteases. An irreversible inhibitor was designed based upon the mechanism and substrate specificity of the target protease. Hosts secreting high amounts of proteases were selected. Impaired hosts were produced by deleting or altering the nucleotide sequence for the proteases.

Streptomyces proteases and improved streptomyces strains for expression

A DNA signal sequence initially isolated from Streptomyces griseus encodes a signal peptide which directs the secretion, via a fused intermediate, of a protein from the cell within which the DNA signal sequence is expressed. The signal sequence is derived from genes encoding protease A and protease B of S. griseus. The DNA signal sequence encodes a thirty-eight amino acid signal peptide. A DNA construct, including the DNA signal sequence and a gene sequence encoding a protein, when transformed into a living cell by a suitable vector, results in the signal peptide correctly directing the secretion of a mature protein of desired structure, particularly from prokaryotic genera selected for their ability to display enzymatic activity of a type typified by, but not exclusive to, that of protein disulphide oxidoreductase, EC 5.3.4.1, more particularly in the genera Streptomyces, and most particularly in Streptomyces lividans 66.

Expression system comprising DNA encoding the signal peptide of protease

The invention relates to a method for the synthesis of ribonucleic acid (RNA) starting from deoxyribonucleic acid (DNA). In the method of the invention, a double stranded DNA molecule is cleaved with a restriction endonuclease so as to generate a free 3'-end. The resultant DNA molecule is then denatured and hybridized with a primer that anneals to the 3'-end of the denatured DNA. The primer used in the hybridization contains a T7 promoter sequence at its 5'-end. Following the hybridization step, the 3'-ends of the resultant hybrid DNA molecule are extended with DNA polymerase to generate a template suitable for T7 RNA polymerase mediated RNA synthesis.

Method for the synthesis of ribonucleic acid (RNA)

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.

This invention relates to an improved process for amplifying a specific nucleic acid sequence. The process involves synthesizing single-stranded RNA, single-stranded DNA and Double-stranded DNA. The single-stranded RNA is a first template for a first primer, the single-stranded DNA is a second template for a second primer, and the double stranded DNA is a third template for synthesis of a plurality of copies of the first template. A sequence of the first primer or the second primer is complementary to a sequence of the specific nucleic acid and a sequence of the first primer or the second primer is homologous to a sequence of the specific nucleic acid. The improvement of the amplification process involves the addition of DMSO alone or in combination with BSA, which improves the specificity and efficiency of the amplification. The amplification process may be used to increase the quantity of a specific nucleic acid sequence to allow detection, or to increase the purity of a specific nucleic acid sequence as a substitute for conventional cloning methodology.

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Brampton, Canada

Lawrence T Malek