Protein Chemical Characteristics of Thermostable Flap Endonuclease from Pyrococcus horikoshii
Satoko KAWASAKI*a,c, Eriko MATSUIa,d, Hiroyasu ISHIDAa, Kazuhiko ISHIKAWAa, Yoshitsugu KOSUGIa, Feng-Wen CUIa, Yutaka KAWARABAYASHIa,b, Ikuo MATSUIa
a National Institute of Bioscience and Human-technology, Higashi 1-1, Tsukuba 305, Japan
b National Institute of Technology and Evaluation, MITI, Nishihara, Shibuyaku, Tokyo, Japan
c New Energy and Industrial Technology Development Organization, Higashi ikebukuro, Toshimaku, Tokyo, Japan
d Japan Tabaco, Aobaku, Yokohama, Kanagawa, Japan
The hyperthermophilic archaeon Pyrococcus horikoshii can grow up to 102℃, and the optimal temperature is 95℃. The genome sequencing project of P. horikoshii is proceeding. The flap endonuclease gene homolog was identified in the genome of P. horikoshii. The flap endonuclease was first purified and characterized in murine and yeast. This enzyme cleaves 5' flap structure endonucleolytically. Recently, it is suggested that this enzyme has a role in replication and repair systems in eucaryotes. Significant homology region in this enzyme is found between murine and yeast, termed N, I and C. So, we have tried to characterize this enzyme in P. horikoshii. This flap endonuclease homologous gene was cloned to two kinds of pET vector and overexpressed in E. coli BL21 (DE3). One was cloned to pET 15b, with a histidine tag at the N-terminal (his-tag type) and another was cloned to pET11 a (native type). The proteins of both types were purified on ion-exchange column. On the process of purification, proteolysis products coexisted in protein solutions. His-tag type protein was finally purified on a heparin column, and proteolysis products were completely removed. We could not completely remove the proteolysis products from the native type protein. This native type protein solution containing proteolysis products was applied to a gel filtration column. But we could not separate these mixture proteins to each single component. The proteolysis sites in this protein were revealed by analysis of N-terminal amino acid sequence in proteolysis products. Some proteolysis sites were between the N and I regions, and between the I and C regions. From these results, these proteolysis sites may be exposed on the surface of this protein. It is suggested that this protein is made up of some units. So, we attempted to construct these units, to analyze the function of this protein.
