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논문 기본 정보
- 자료유형
- 학술저널
- 저자정보
- 저널정보
- 한국미생물생명공학회 Journal of Microbiology and Biotechnology Journal of Microbiology and Biotechnology 제20권 제3호
- 발행연도
- 2010.1
- 수록면
- 542 - 549 (8page)
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초록· 키워드
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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 vector pET21a. 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 90oC 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 on temperature. In the presence of ATP, ApCpnB effectively protected citrate synthase (CS) and alcohol dehydrogenase (ADH) from thermal aggregation and inactivation at 43oC and 50oC, respectively. Specifically, the activity of malate dehydrogenase (MDH) at 85oC was greatly stabilized by the addition of ApCpnB and ATP. Coexpression of procarboxypeptidase 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 a powerful molecular machinery for the production of recombinant proteins as soluble and active forms in E. coli.
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참고문헌 (23)
참고문헌 신청
Andra, S / 1996 / Purification and structural characterization of the thermosome from the hyperthermophilic archaeum Methanopyrus kandleri / FEBS Lett 379 : 127 ~ 131
Andra, S / 1998 / The thermosome from Methanopyrus kandleri possesses an NH4 +- dependent ATPase activity / Eur. J. Biochem 255 : 93 ~ 99
Archibald, J. M / 1999 / Recurrent paralogy in the evolution of archaeal chaperonins / Curr. Opin. Biotechnol 9 : 1053 ~ 1056
Buchner, J / 1991 / GroE facilitates refolding of citrate synthase by suppressing aggregation / Biochemistry 30 : 1586 ~ 1591
Bult, C. J / 1996 / Complete genome sequence of the methanogenic archaeon Methanococcus jannaschii / Science 273 : 1058 ~ 1073
Andra, S / 1998 / The thermosome from Methanopyrus kandleri possesses an NH4 +- dependent ATPase activity / Eur. J. Biochem 255 : 93 ~ 99
Archibald, J. M / 1999 / Recurrent paralogy in the evolution of archaeal chaperonins / Curr. Opin. Biotechnol 9 : 1053 ~ 1056
Buchner, J / 1991 / GroE facilitates refolding of citrate synthase by suppressing aggregation / Biochemistry 30 : 1586 ~ 1591
Bult, C. J / 1996 / Complete genome sequence of the methanogenic archaeon Methanococcus jannaschii / Science 273 : 1058 ~ 1073
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