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Overexpression, purification, and functional characterization of the group II chaperonin from the hyperthermophilic archaeum Pyrococcus horikoshii OT3

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Abstract

Overexpression in Escherichia coli and functional characterization of the group II chaperonin from the hyperthermophilic archaeum Pyrococcus horikoshii OT3 were investigated in this study. PhCpn, the chaperonin gene from the P. horikoshii OT3, was amplified by PCR from the P. horikoshii genomic DNA, subcloned into pET21a vector, and expressed in three E. coli host cells such as BL21, Rosetta, and Codonplus (DE3). Among these host cells, E. coli Rosetta showed the highest expression level of recombinant PhCpn at induction with 1 mM IPTG. The recombinant PhCpn was purified to 91% by heat-shock treatment and anion-exchange chromatography. The ATPase activity of the purified PhCpn increased in a PhCpn concentration-dependent manner. Also, PhCpn protected the inorganic phosphatase from thermal inactivation at 85 and 110°C, speculating that PhCpn is effective in in vitro holding of the protein. The holding efficiency was enhanced by the addition of Mg2+ ion. Through the coexpression of pro-carboxypeptidase B (pro-CPB) and PhCpn in E. coli Rosetta, pro-CPB was produced as a soluble and active form with a marked yield. This result indicated that PhCpn facilitated the in vivo correct folding of pro-CPB and could be used as powerful and novel molecular machinery for the production of recombinant proteins as soluble and active forms in E. coli.

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Authors and Affiliations

  1. Department of Biomaterial Control, Dong-Eui University, Busan, 614-714, Korea

    Jeong-Hwan Kim, Eun-Jung Shin, Sung-Jong Jeon, Yeon-Hee Kim & Soo-Wan Nam

  2. Department of Biotechnology and Bioengineering, Dong-Eui University, Busan, 614-714, Korea

    Sung-Jong Jeon, Yeon-Hee Kim & Soo-Wan Nam

  3. Department of Biotechnology, Catholic University of Korea, Gyunggi, 420-743, Korea

    Pil Kim

  4. Department of Bioscience and Biotechnology, Konkuk University, Seoul, 143-701, Korea

    Chung-Hwan Lee

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Kim, JH., Shin, EJ., Jeon, SJ. et al. Overexpression, purification, and functional characterization of the group II chaperonin from the hyperthermophilic archaeum Pyrococcus horikoshii OT3. Biotechnol Bioproc E 14, 551–558 (2009). https://doi.org/10.1007/s12257-009-0008-0

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