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KMID : 0545120100200030542
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Journal of Microbiology and Biotechnology
2010 Volume.20 No. 3 p.542 ~ p.549
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Overexpression, Purification, and Characterization of β-Subunit Group II Chaperonin from Hyperthermophilic Aeropyrum pernix K1
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Shin Eun-Jung
Jeon Seong-Soo
Kim Yeon-Hee
Nam Soo-Wan
Kim Jeong-Hwan
Lee Jin-Woo
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Abstract
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In the present study, overexpression, purification, and characterization of Aeropyrum pernix K1 chaperonin B in E. coli were investigated. The chaperonin β-subunit gene (ApCpnB, 1,665 bp ORF) from the hyperthermophilic archaeon A. pernix K1 was amplified by PCR and subcloned into pET21a vector. The constructed pET21a-ApCpnB (6.9 kb) was transformed into E. coli BL21 Codonplus (DE3). The transformant cell successfully expressed ApCpnB, and the expression of ApCpnB (61.2 kDa) was identified through analysis of the fractions by SDS-PAGE (14% gel). The recombinant ApCpnB was purified to higher than 94% by using heat-shock treatment at 90?C for 20 min and fast protein liquid chromatography on a HiTrap Q column step. The purified ApCpnB showed ATPase activity and its activity was dependent with temperature. In the presence of ATP, ApCpnB effectively protects citrate synthase (CS) and alcohol dehydrogenase (ADH) from thermal aggregation and inactivation at 43?C and 50?C, respectively. Especially, the activity of malate dehydrogenase (MDH) at 85?C was greatly stabilized by the addition of ApCpnB and ATP. Coexpression of pro-carboxypeptidase B (pro-CPB) and ApCpnB in E. coli BL21 Codonplus (DE3) had a marked effect on the yield of pro-CPB as a soluble and active form, speculating that ApCpnB facilitates the correct folding of pro-CPB. These results suggest that ApCpnB has both foldase and holdase activities and can be used as powerful molecular machinery for the production of recombinant proteins as soluble and active forms in E. coli.
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KEYWORD
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Chaperonin, Aeropyrum pernix, ATPase activity, citrate synthase, malate dehydrogenase, pro-carboxypeptidase B
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