ORIGINAL PAPER Enrichment and proteome analysis of a hyperthermostable protein set of archaeon Thermococcus onnurineus NA1 Sung-Ho Yun • Chi-Won Choi • Sang Oh Kwon • Yeol Gyun Lee • Young-Ho Chung • Hoi Jong Jung • Yun-Jae Kim • Jung-Hyun Lee • Jong-Soon Choi • Soohyun Kim • Seung Il Kim Received: 4 September 2010 / Accepted: 6 April 2011 / Published online: 23 April 2011 Ó Springer 2011 Abstract Thermococcus onnurineus NA1 is a hyper- thermophilic archaeon that can be used for the screening of thermophilic enzymes. Previously, we characterized the metabolic enzymes of the cytosolic proteome by two- dimensional electrophoresis/tandem mass spectrometry (2-DE/MS–MS). In this study, we identified a subset of hyperthermostable proteins in the cytosolic proteome using enrichment by in vitro heat treatment and protein identifi- cation. After heat treatment at 100°C for 2 h, 13 and 149 proteins were identified from the soluble proteome subset by 2-DE/MS–MS and 1-DE/MS–MS analysis, respectively. Representative proteins included intracellular protease I, thioredoxin reductase, triosephosphate isomerase, putative hydroperoxide reductase, proteasome, and translation initiation factors. Intracellular protease, deblocking ami- nopeptidases, and fructose-1,6-bisphosphatase were over- expressed in Escherichia coli and biological activity above 85°C was confirmed. The folding transition temperature (Tm) of identified proteins was analyzed using the in silico prediction program TargetStar. The proteins enriched with the heat treatment have higher Tm than the homologous proteins from mesophilic strains. These results suggested that the heat-stable protein set of hyperthermophilic T. onnurineus NA1 can be effectively fractionated and enriched by in vitro heat treatment. Keywords Archaea Á Thermococcus Á Hyperthermostable Á Proteome Introduction Hyperthermophiles are valuable living things for the elu- cidation of adaptation mechanism of microorganisms inhabiting in extreme environment. Recently, proteomic approaches have been applied to understand metabolic characteristics of hyperthermophiles. Proteomic mining has been used to comprehensively investigate the proteins of hyperthermophiles (Barry et al. 2006; Burghardt et al. 2008; Kwon et al. 2009; Lee et al. 2008a; Yamazaki et al. 2006; Wang et al. 2004). These results revealed the ele- mental metabolic characters of the strains. In the case of Thermoanaerobacter tengcongensis and Thermus thermo- philus, comparative proteome analysis was performed to identify proteins associated with hyperthermophilic activ- ity by using the strains cultured in different temperatures (Trauger et al. 2008; Wang et al. 2007); the results revealed which proteins were up- and downregulated according to stimulation by temperature of culture. However, proteomic approaches to screen heat-stable proteins of hyperthermo- philes was not intensively performed. In recent, thermal and chemical perturbation methods have been used for screening of thermostable proteome (Prosinecki et al. 2006). This method lowered the complexity of the soluble Communicated by F. Robb. S.-H. Yun and C.-W. Choi contributed equally to this work. Electronic supplementary material The online version of this article (doi:10.1007/s00792-011-0376-1) contains supplementary material, which is available to authorized users. S.-H. Yun Á C.-W. Choi Á S. O. Kwon Á Y. G. Lee Á Y.-H. Chung Á J.-S. Choi Á S. Kim Á S. I. Kim (&) Division of Life Science, Korea Basic Science Institute, Daejeon 305-333, Korea e-mail: ksi@kbsi.re.kr H. J. Jung Á Y.-J. Kim Á J.-H. Lee Korea Ocean Research & Development Institute, P.O. Box 29, Ansan, Seoul 425-600, Korea 123 Extremophiles (2011) 15:451–461 DOI 10.1007/s00792-011-0376-1