• 대한수의학회지
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• 제45권1호
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• pp.45-53
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• 2005

GroE that is a heat shock protein composed of GroEL and GroES is known as an immunodominant target of both the humoral and cellular immune responses in bovine brucellosis. This study was carried out to characterize groE gene encoding heat shock proteins of B. abortus isolated in Korea and to evaluate the immunogenicity of the GroE protein expressed in E. coli system. In PCR the specific signals with the size of 2,077 bp were detected in five strains isolated from the mammary lymphnodes of the dairy cattle that were serologically positive and the reference strains. In comparison of the sequences of nucleotides and amino acids among the strains, GroES showed 100% identity in both sequences. GroEL was evaluated 99.0~99.9% in nucleotides and 98.0~100% homology in amino acids. The groE gene including groES and groEL was inserted into pET29a vector and constructed pET29a-GroE recombinant plasmids. The inserted groE was confirmed by digestion with Nco1 and EcoR1 endonucleases and nucleotide sequencing. E. coli BL (DE3) was transformed with pET29a-GroE, named as E. coli BL (DE3)/pET29a-GroE. In SDS-PAGE, it was evident that the recombinant plasmid effectively expressed the polypeptides for GroES (10 kDa) and GroEL (60 kDa) in 0.5, 1 and 2 hours after IPTG induction. The immuno-reactivity of the expressed proteins were proved in mouse inoculation and Western blot analysis.

• 미생물학회지
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• 제32권1호
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• pp.47-52
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• 1994

Campylobacter jejuni에 48${\circ}C$의 열충격을 30분간 주었을 때, HSP90, HSP66, HSP60의 열충격 단백질들이 합성되었고, 이 단백질들은 각각 E. coli의 hsp87, HSP66 (DnaK), HSP58(GroEL)에 상응하는 단백질들이었다. 여러가지의 제한효소로 처리한 C. jejuni의 chromosomal DNA에 E. coli의 groEL(4.0kb)을 probe로 사용하여 Southern hybridization한 결과, 이들과 상동성을 가지는 유전자들이 있음을 확인하였다. C. jejuni의 groEL 유전자를 pWE15 cosmid를 이용하여 recombinant plasmid pLC1을 만들고, 이를 E. coli B178 groEL44 ts mutant에 형질전환시켜 E. coli LC1을 얻었다. 이 pLC1에는 groEL 유전자가 존재하는 5.7kb인 insert DNA가 포함되어 있었고, 그로부터 subcloning한 pLC101에는 groEL을 포함하는 4.0kb의 DNA가 삽입되어 있었다. 이 recombinant plasmid들이 형질전환된 E. coli LC1과 LC101 균주에서는 C. jejuni의 GroEL 단백질이 과다 생산되었다. C. jejuni의 groEL이 cloning된 E. coli LC1은 42${\circ}C$에서의 생장능력이 회복되었고, ${\lambda}$ vir phage에 대한 감수성도 회복되는 등의 chaperon 효과가 입증되었다.

• Journal of Microbiology
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• 제38권3호
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• pp.145-149
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• 2000

GroEL is a typical molecular chaperone. GroEL synthesis patterns at various culture temperatures in Escherichia coli were investigated in this study. No significant differences in the amount of GroEL produced from the chromosome were found at 30 and 37$^{\circ}C$. However, GroEL production increased 3.4-fold at 42$^{\circ}C$. GroEL synthesis was not transient but continuous at 42$^{\circ}C$, although most heat shock gene expression is known to be transient. To understand the role of the groEL structural gene, a groE promoter-lacZ fusion was constructed. Interestingly , while transcriptional fusion is not thermally inducible, it is inducible by ethanol, suggesting that the secondary structure of the groEL transcript is involved in thermal regulation of the groEL gene. Secondary structures of groE mRNA at 37 and 42$^{\circ}C$ were compared using the computer program RNAdraw. Distinct structures at the two temperatures were found, and these structures may be related to a high level of GroEL expression at 42$^{\circ}C$.

• Journal of Microbiology and Biotechnology
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• 제10권1호
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• pp.63-68
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• 2000

GroEL is well known as a molecular chaperone. In order to determine the dynamic stress response of the Escherichia coli groE promoter, a groE-lacZ operon fusion in the chromosome was constructed. Stress leading to ${\sigma}^{32}$ synthesis induces transcription from E. coli groE promoter, since the promoter is ${\sigma}^{32}-regulated$. When the strain was stressed with ethanol, phenol, and sodium chloride, clear inductions of ${\beta}-galactosidase$ were observed. Two types of simultaneous stresses of sodium chloride and phenol induced the enze much more than either of the two alone, suggesting that stress was an additive. The combined stress resulted in the highest induction of the enzyme in this system. The groE-lacZ fusion strain developed in this study can conveniently be used to detect other harmful pollutants in the environment. Stress treatment of cells containing recombinant proteins, which need GroEl, by ethanol, phenol, or sodium chloride, might have a tendency to increase their biological activities.

• Journal of Microbiology
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• 제34권1호
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• pp.15-22
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• 1996

The cloned X-bacterial gene (groEx) which is analogous to groE of E. Coli strongly expressed in E. coli when grown at the temperature 27.deg. C or higher without having to add any inducers. By S1-nuclease mapping, primer extension analysis and site directed mutagenesis, we found 4 promoters in the gene. Among them two promoters located at 5'-extended region of the gene are homologous to the promoters found in groE family of heat-shock genes ; they are , .sigma.$^{32}$ factor-dependent P1 promotor and .delta$^{70}$factor-dependet P2 promoter. The other two promoters found within the coding region of groESx were P3, 5'-TTGGCG-(18 bases)-AATACT-3' and P4, 5'-TTGGCA-(19 bases)-TAAGT which overlapped within 49 bases. These unique intragenic .delta.$^{70}$-dependent promoters are the first to be cloned and characterized in groE analogous heat-shock genes so far. These P3 and P4 promoters appeared to be responsible for the strong expression of GroElx in X-bacteria in vivo.

• Journal of Microbiology and Biotechnology
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• 제26권2호
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• pp.337-346
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• 2016

Three combinations of molecular chaperones from Escherichia coli (i.e., DnaK-DnaJ-GrpE-GroEL-GroES, GroEL-GroES, and DnaK-DnaJ-GrpE) were overproduced in E. coli BL21, and their in vitro stabilizing effects on a nitrile hydratase (NHase) were assessed. The optimal gene combination, E. coli groEL-groES (ecgroEL-ES), was introduced into Rhodococcus ruber TH3. A novel engineered strain, R. ruber TH3G was constructed with the native NHase gene on its chromosome and the heterologous ecgroEL-ES genes in a shuttle plasmid. In R. ruber TH3G, NHase activity was enhanced 37.3% compared with the control, TH3. The in vivo stabilizing effect of ecGroEL-ES on the NHase was assessed using both acrylamide immersion and heat shock experiments. The inactivation behavior of the in vivo NHase after immersion in a solution of dynamically increased concentrations of acrylamide was particularly evident. When the acrylamide concentration was increased to 500 g/l (50%), the remaining NHase activity in TH3G was 38%, but in TH3, activity was reduced to 10%. Reactivation of the in vivo NHases after varying degrees of inactivation was further assessed. The activity of the reactivated NHase was more than 2-fold greater in TH3G than in TH3. The hydration synthesis of acrylamide catalyzed by the in vivo NHase was performed with continuous acrylonitrile feeding. The final concentration of acrylamide was 640 g/l when catalyzed by TH3G, compared with 490 g/l acrylamide by TH3. This study is the first to show that the chaperones ecGroEL-ES work well in Rhodococcus and simultaneously possess protein-folding assistance functions and the ability to stabilize and reactivate the native NHases.

• KSBB Journal
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• 제23권5호
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• pp.403-407
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• 2008

본 연구에서는 현재 산업적 응용이 활발하게 이루어지고 있고, 여러 장점을 지닌 효소인 Candida antarctica에서 유래된 lipase B (CalB)의 신속한 개질을 위해 취급이 용이한 E. coli를 이용하여 CalB 발현시스템을 구축하였다. E. coli 발현 시스템에서 효소활성을 지니지 못하는 내포체를 생성하는 단점을 지니고 있어, soluble한 형태의 CalB 생성을 위해 저온 발현이 가능한 pCold I vector와 단백질 접힘을 도와주는 chaperone을 사용하여 CalB를 발현하였다. Liu 등(17)은 E. coli Origami2와 B, 그리고 $DH5{\alpha}$를 실험한 결과, Origami 균주에서만 CalB에 의한 halo의 형성이 관찰되었으나, 동 연구에서는 실험한 3종의 균주와 5종의 chaperone plasmid중 Rosettagami와 $DH5{\alpha}$에서 groES/groEL chaperone이 CalB와 동시에 발현되면 soluble한 형태의 Cal B가 발현됨을 관찰할 수 있었다. 또한 신속한 CalB의 발현시스템을 구축하기 위해서는 유전자 조작의 용이성 및 안정성에서 우월한 $DH5{\alpha}$가 Rosettagami에 비해 soluble한 CalB의 발현에 더욱 적합한 균주임이 관찰되었다. 즉 재조합 pCold plasmid와 pGro7 plasmid (groES/groEL)로 형질이 전환된 $DH5{\alpha}$가 CalB 발현시스템에 가장 적합하다.

• KSBB Journal
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• 제14권4호
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• pp.429-433
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• 1999

$\beta$-Glucosidaes from Cellvibrio gilvus(CG) was successfully overproduced in soluble form in E. coli with the coexpression of GroEL/ES/. Without the GroEL/ES protein, the $\beta$-glucosidase overexpressed in E. coli constituted a huge amount(80%) of total cellular protein, but was localized in the insoluble fraction, and little activity was detected in the soluble fraction. Coexpression of the E. coli GroEL/ES had a drastic impact on the proper folding of the $\beta$-glucosidase; 20% of the overexpressed enzyme was recovered in the soluble fraction in active form. Similar effects of GroEL/ES were also observed on the overexpressed $\beta$-glucosidase from Agrobacterium tumefaciens(AT). And pET28(a)-RGRAR, partially deleted mutant lacking 5-amino acid residues at carboxy teminus also could be folded into an active form when expressed with the molecular chaperonin GroEL/ES, and its activity was higher than that of the without GroEL/ES system, In addition, the synergistic effect of GroEL/ES and the low induction temperature were important factors for solubilization of the inclusion body from overproduced $\beta$-glucosidases.

• 생명과학회지
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• 제12권6호
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• pp.688-693
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• 2002

Chaperone 분자는 세포 내에서 새로 합성된 polypeptides의 misfolding을 보호하는 역할을 가진다. 이런 chaperone 분자와의 공발현은 활성형 재조합 단백질의 생산을 증가를 기대할 수 있다. 본 연구에서는 E. cozi에서 B. macerans 유래 cyclodextrin glucanotransferase (CGTase)의 활성형 생산에 GroEL/ES chaperone과의 공발현의 효과에 대해 조사하였다. cgt와 groEL/ES 유전자출 발현하는 pTCGT1과 pGro7은 각각 T7 promoter와 araB promoter에 의해 조절되고 이들을 E. coli cell에 co-transformation시켰다. 재조합 E. coli에서 IPTG와 L-arabinose의 최적 농도를 결정하기 위해 행한 결과 1 mM IPTG, 0.3 mg L-arabinose/$m\ell$에서 가장 높은 CGTase 활성을 나타내었다. 그리고 tube에서는 L-arabinose와 IPTG를 각각 0.4~0.5 $OD_{600}$과 0.8~l.0 $OD_{600}$에서 첨가하였을 때 활성형 CGTase의 생산이 증가되었다. GroEL/ES 공발현 조건에서는 가용성 CGTase 활성이 0.7~0.73 unit/$m\ell$로 단독 발현의 0.36~0.56 unit/$m\ell$에 비해 약 1.5 배 정도 증가함을 알 수 있었다. SDS-PAGE 분석에서는 GroEL/ES 공발현 조건에서 총 CGTase의 33.6%정도가 가용성 형태로 생산됨을 알 수 있었다.

• International Journal of Oral Biology
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• 제37권3호
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• pp.130-136
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• 2012

The GroEL heat-shock protein from Fusobacterium nucleatum, a periodontopathogen, activates risk factors for atherosclerosis in human microvascular endothelial cells (HMEC-1) and ApoE-/- mice. In this study, we analyzed the signaling pathways by which F. nucleatum GroEL induces the proinflammatory factors in HMEC-1 cells known to be risk factors associated with the development of atherosclerosis and identified the cellular receptor used by GroEL. The MAPK and NF-${\kappa}B$ signaling pathways were found to be activated by GroEL to induce the expression of interleukin-8 (IL-8), monocyte chemoattractant protein 1 (MCP-1), intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), E-selectin, and tissue factor (TF). These effects were inhibited by a TLR4 knockdown. Our results thus indicate that TLR4 is a key receptor that mediates the interaction of F. nucleatum GroEL with HMEC-1 cells and subsequently induces an inflammatory response via the MAPK and NF-${\kappa}B$ pathways.