Overexpression of RNA polymerase subunits in extremely halophilic archaeon, Haloarcula japonica and its subunit-subunit interaction analyses

概要

論文の詳細を見る
All subunit genes (rpoA, B1, B2, C, D, E1, E2, F, M1, M2, L, H, N, K and P) encoding for RNA polymerase in extremely halophilic archaeon, Haloarcula japonica, were cloned, sequenced, and their structural analyses were carried out. It encodes 15 subunit genes which are separately located on the chromosome. These genes were then cloned into overexpression plasmid, pCold I which is used for its overproduction of fusion protein with N-terminal hexahistidine tag in a cold shock condition. These plasmids for overexpression of all subunits of H. japonica RNA polymerase, were expressed in a Escherichia coli JM109 host and the expressed proteins were analyzed by SDS-PAGE. Out of 15 plasmids, 13 (RpoA, B1, B2, C, D, E1, E2, M1, M2, L, H, N and P) subunits were successfully overexpressed and two (RpoK and F) were failed. In addition, construction of bait and target plasmid sets were performed. These were then used for subunit-subunit interaction analyses using BacterioMatch II two hybrid system. The analyses confirmed strength in subunit-subunit interactions of H. japonica RNA polymerase. These results together give us the technique of in vitro reconstitution of H. japonica RNA polymerase.
近畿大学の論文