PSEUDO-AFFINITY COLUMN CHROMATOGRAPHY BASED RAPID PURIFICATION PROCEDURE FOR T7 RNA-POLYMERASE

Based on the observation that T7 RNAP binds reversibly to the polyarom atic sulphonated triazine dye, cibacron blue, we have developed a rapi d 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 elut ed by 2M NaCl or 1M NaCl together with 1 mM UTP. These methods are com pared with another purification procedure using ion-exchange column ch romatography. All of them yield essentially pure T7 RNAP with high spe cific activity.