Abstract
Although RNA polymerase purification methods have been reported in a number of cyanobacterial species, they are very complicated and time-consuming.Here we report a simple and rapid purification of RNA polymerase from Synechocystis sp. strain PCC 6803, by using a carboxy terminal His6-tagged RpoC2 (beta' subunit) mutant strain. A vector for chromosomal displacement was made by placing the Cm cassette immediately downstream of the rpoC2-(CAC)6 construct. The generated mutant strain grew as well as the wild type. For purification, cells were disrupted using glass beads, and the resulting high speed supernatant (20 mM Tris-Cl pH 8, 0.5 M NaCl, 5% glycerol) was loaded on a Ni-NTA column. Western analysis revealed that the 20-50 mM eluate contained the subunits of RNA polymerase (beta', beta, gamma, alpha, sigma). We also describe the results of in vitro transcription analysis using the purified enzyme.
