• Bulletin of the Korean Chemical Society
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• 제30권12호
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• pp.3143-3146
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• 2009

• Journal of Microbiology and Biotechnology
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• 제20권4호
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• pp.727-731
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• 2010

The gene APE0743 encoding the superoxide dismutase (ApSOD) of a hyperthermophilic archaeon Aeropyrum pernix K1 was cloned and overexpressed as a GST fusion protein at a high level in Escherichia coli. The expressed protein was simply purified by the process of glutathione affinity chromatography and thrombin treatment. The ApSOD was a homodimer of 25 kDa subunits and a cambialistic SOD, which was active with either Fe(II) or Mn(II) as a cofactor. The ApSOD was highly stable against high temperature. This thermostable ApSOD is expected to be applicable as a useful biocatalyst for medicine and bioindustrial processes.

• Journal of Microbiology and Biotechnology
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• 제21권2호
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• pp.212-217
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• 2011

${\alpha}$ and ${\beta}$-subunits (ApCpnA and ApCpnB) are group II chaperonins from the hyperthermophilic archaeum Aeropyrum pernix K1, specialized in preventing the aggregation and inactivation of substrate proteins under conditions of transient heat stress. In the present study, the cooperativity of ${\alpha}$- and ${\beta}$-subunits from the A. pernix K1 was investigated. The ApCpnA and ApCpnB chaperonin genes were overexpressed in E. coli Rosetta and Codonplus (DE3), respectively. Each of the recombinant ${\alpha}$- and ${\beta}$-subunits was purified to 92% and 94% by using anionexchange chromatography. The cooperative activity between purified ${\alpha}$- and ${\beta}$-subunits was examined using citrate synthase (CS), alcohol dehydrogenase (ADH), and malate dehydrogenase (MDH) as substrate proteins. The addition of both ${\alpha}$- and ${\beta}$-subunits could effectively protect CS and ADH from thermal aggregation and inactivation at $43^{\circ}C$ and $50^{\circ}C$, respectively, and MDH from thermal inactivation at $80^{\circ}C$C and $85^{\circ}C$. Moreover, in the presence of ATP, the protective effects of ${\alpha}$- and ${\beta}$-subunits on CS from thermal aggregation and inactivation, and ADH from thermal aggregation, were more enhanced, whereas cooperation between chaperonins and ATP in protection activity on ADH and MDH (at $85^{\circ}C$) from thermal inactivation was not observed. Specifically, the presence of both ${\alpha}$- and ${\beta}$- subunits could effectively protect MDH from thermal inactivation at $80^{\circ}C$ in an ATP-dependent manner.

• Journal of Microbiology and Biotechnology
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• 제20권3호
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• pp.542-549
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• 2010

In the present study, overexpression, purification, and characterization of Aeropyrum pernix K1 chaperonin B in E. coli were investigated. The chaperonin $\beta$-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 $90^{\circ}C$ 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 on temperature. In the presence of ATP, ApCpnB effectively protected citrate synthase (CS) and alcohol dehydrogenase (ADH) from thermal aggregation and inactivation at $43^{\circ}$ and $50^{\circ}$, respectively. Specifically, the activity of malate dehydrogenase (MDH) at $85^{\circ}$ 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 a powerful molecular machinery for the production of recombinant proteins as soluble and active forms in E. coli.

• Bulletin of the Korean Chemical Society
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• 제28권12호
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• pp.2261-2265
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• 2007

Prevention of thermal aggregation of the denatured protein by the group II chaperonin from the aerobic hyperthermophilic crenarchaeon Aeropyrum pernix K1 (ApcpnA) has been investigated. ApcpnA exists as a homo-oligomer in a ring structure, which protects thermal aggregation of the chemically denatured bovine rhodanese at 50 oC. ApcpnA alone is not sufficient for chaperonin activity, but the chaperonin activity is greatly enhanced in the presence of manganese ion and ATP. Compared to the mesophilic chaperonin GroEL/GroES, ApcpnA is more activated at a higher temperature and protects the aggregation-prone unfolded state of the denatured rhodanese from thermal aggregation. Binding of ATP is sufficient for ApcpnA to perform the chaperonin function in vitro, but hydrolysis of ATP is not necessarily required. We propose that utilization of Mn2+ and adenosine nucleotide regardless of ATP hydrolysis may be one of peculiar properties of archaeal chaperonins.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2005년도 생물공학의 동향(XVI)
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• pp.386-386
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• 2005

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2005년도 생물공학의 동향(XVII)
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• pp.492-492
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• 2005

• 한국생명과학회:학술대회논문집
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• 한국생명과학회 2006년도 제47회 학술심포지움 및 추계국제학술대회
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• pp.91.2-91.2
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• 2006

• Journal of Microbiology and Biotechnology
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• 제23권6호
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• pp.818-825
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• 2013

We isolated and functionally characterized the ${\alpha}$- and ${\beta}$-subunits (ApCpnA and ApCpnB) of a chaperonin from Aeropyrum pernix K1. The constructed vectors pET3d-ApCpnA and pET21a-ApCpnB were transformed into E. coli Rosetta (DE3), BL21 (DE3), or CodonPlus (DE3) cells. The expression of ApCpnA (60.7 kDa) and ApCpnB (61.2 kDa) was confirmed by SDS-PAGE analysis. Recombinant ApCpnA and ApCpnB were purified by heat-shock treatment and anion-exchange chromatography. ApCpnA and ApCpnB were able to hydrolyze not only ATP, but also CTP, GTP, and UTP, albeit with different efficacies. Purified ApCpnA and ApCpnB showed the highest ATPase, CTPase, UTPase, and GTPase activities at $80^{\circ}C$. Furthermore, the addition of ApCpnA and ApCpnB effectively protected citrate synthase (CS) and alcohol dehydrogenase (ADH) from thermal aggregation and inactivation at $43^{\circ}C$ and $50^{\circ}C$, respectively. In particular, the addition of ATP or CTP to ApCpnA and ApCpnB resulted in the most effective prevention of thermal aggregation and inactivation of CS and ADH. The ATPase activity of the two chaperonin subunits was dependent on the salt concentration. Among the ions we examined, potassium ions were the most effective at enhancing the ATP hydrolysis activity of ApCpnA and ApCpnB.

• 생명과학회지
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• 제25권2호
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• pp.130-135
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• 2015

Pseudoalteromonas elyakovii 유래 alginate lyase 유전자(aly)를 대장균에서 발현시켰을 때, 발현된 대부분의 유전자 산물은 내포체라는 불용성 응집체 형태로 생산되었다. Alginate lyase를 가용성 및 활성형으로 생산하기 위해 Aeropyrum pernix K1 유래의 초고온성 샤페로닌 ApCpnA와 ApCpnB를 공발현 파트너로 도입하였다. aly와 ApCpnA와 ApCpnB 각각과의 공발현 결과, aly 단독발현 때의 alginate lyase 활성 10.1 unit/g-soluble protein에서 ApCpnA와의 공발현 때는 83.1 unit/g-soluble protein, ApCpnB와의 공발현 때는 100.3 unit/g-soluble protein으로 가용성 및 활성형으로의 alginate lyase 생산이 8~10배 크게 향상되었다. 다양한 배양 조건들의 조사를 통해 alginate lyase 최대 생산을 위한 조건은 다음과 같았다: 1.0 mM IPTG, $25^{\circ}C$ 배양 온도(IPTG 유도 후), ApCpnB 공발현 파트너. 이러한 공발현 시스템은 대장균에서 기능적으로 또한 의학적으로 중요한 재조합 단백질의 산업적 생산에 크게 유용하게 사용될 것이다.