摘要 |
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 <i>E. coli</i> 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 <i>E. coli</i> 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 <i>in vivo</i> system to express RNAs and proteins under mini vRNA polymerase promoter control. |
申请人 |
UNIVERSITY OF CHICAGO;KAZMIERCZAK, KRYSTYNA, M.;DAVYDOVA, ELENA, K.;ROTHMAN-DENES, LUCIA, B. |
发明人 |
KAZMIERCZAK, KRYSTYNA, M.;DAVYDOVA, ELENA, K.;ROTHMAN-DENES, LUCIA, B. |