N4 virion single-stranded dna dependent rna polymerase

Details N4 virion single-stranded dna dependent rna polymerase N4 virion single-stranded dna dependent rna polymerase

C - Chemistry, Metallurgy

12

N

C12N 15/54 (2006.01) C07H 21/04 (2006.01) C07K 1/00 (2006.01) C12N 1/20 (2006.01) C12N 1/21 (2006.01) C12N 9/00 (2006.01) C12N 9/12 (2006.01) C12N 15/00 (2006.01) C12N 15/64 (2006.01) C12P 19/34 (2006.01) C12P 21/02 (2006.01) C12Q 1/48 (2006.01) C12Q 1/68 (2006.01)

Patent

CA 2448097

A histidine-tagged, deletion mutant of bacteriophage N4-coded, virion RNA polymerase (mini-vRNAP) which is active has been developed. The his-tagged mini-vRNAP has been cloned under the control of the Pbad promoter, is stable and is purified in a single step yielding large amounts (10 mg/liter of E. coli expressing cells). This RNA polymerase uses single-stranded DNA containing 17 bases (the promoter) upstream of the transcribed regions as a template. In the presence of E. coli SSB protein, it transcribes this template efficiently, providing a unique system to synthesize RNAs of the desired sequence using single-stranded DNA templates. The enzyme incorporates derivatized nucleoside triphosphates with high efficiency. A mutant of mini- vRNAP has been generated that incorporates deoxynucleoside triphosphates. In addition, the inventors have developed an in vivo system to express RNAs and proteins under mini vRNA polymerase promoter control.

L'invention concerne un mutant de délection actif à étiquette d'histidine d'ARN polymérase de virion bactériophage codée N4 (mini-vPARN). Cette mini-vARN polymérase a été clonée sous le contrôle du promoteur pBAD, est stable et a été purifiée en une seule étape de manière à produire des quantités importantes (10 mg/litre de cellules exprimant <i>E. coli</i>). Cette ARN polymérase utilise comme modèle un ADN à brin unique contenant 17 bases (le promoteur) en amont des régions transcrites. En présence d'une protéine SSB de <i>E. coli</i>, elle transcrit efficacement cette matrice de manière à créer un système unique pour synthétiser des ARN de la séquence désirée en utilisant des matrices d'ADN à brin unique. L'enzyme comprend des nucléoside triphosphates dérivés hautement efficaces. Un mutant de mini-vRNAP a été généré; il comprend des désoxynucléoside triphosphates. En outre, les inventeurs ont développé un système in vivo pour exprimer les ARN et les protéines sous le contrôle des promoteurs de mini-vRNAP.

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