Pseudo-affinity column chromatography based rapid purification procedure for T7 RNA polymerase.
Based on the observation that T7 RNAP binds reversibly to the polyaromatic sulphonated triazine dye, cibacron blue, we have developed a rapid purification procedure for T7 RNAP. It employs chromatography of the ammonium sulfate fraction through a blue sepharose column, which has the dye coupled to the solid sepharose support. The enzyme can be eluted by 2M NaCl or 1M NaCl together with 1 mM UTP. These methods are compared with another purification procedure using ion-exchange column chromatography. All of them yield essentially pure T7 RNAP with high specific activity.
Das
M
M
Biophysics Division, Saha Institute of Nuclear Physics, Calcutta, India.
Dasgupta
D
D
D002846:Chromatography, Affinity / Q000379:methods*; D002852:Chromatography, Ion Exchange; D004396:Coloring Agents / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D014227:Triazines / Q000378:metabolism; D014764:Viral Proteins
D004396:Coloring Agents; D014227:Triazines; D014764:Viral Proteins; C012842:Cibacron Blue F 3GA; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases