• 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.

• 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.

• Journal of Microbiology and Biotechnology
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• 제14권1호
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• pp.216-219
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• 2004

The effect of culture temperature on the production of soluble form of B. macerans cyclodextrin glucanotransferase (CGTase) in recombinant E. coli was investigated. E. coli cell was cotransformed with two plasmids (pTCGT1 and pGroll) in which the cgt and groEL/ES genes are under the control of T7 promoter and pzt-1 promoter, respectively. When tetracycline (10 ng/ml) and IPTG (l mM) were added as inducers at the early-exponential phase (2 h) and mid-exponential phase (3h), respectively, the solubilization of the inclusion body CGTase was greatly dependent on the temperature of the culture. At low culture temperature of $25^\circ{C}$, 2- or 3-fold higher activity and specific activity were obtained over $37^\circ{C}$. SDS-PAGE analysis revealed that about 62% of CGTase in the total CGTase protein was found in the soluble fraction by applying overexpression of GroEL/ES chaperone and by cultivation of E. coli at $25^\circ{C}$, whereas 33% of CGTase was detected in the soluble fraction at $37^\circ{C}$. Therefore, the expression of GroEL/ES and cultivation at $25^\circ{C}$ greatly enhanced the soluble production of CGTase in E. coli.

• 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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• 제12권6호
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• pp.1002-1005
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• 2002

The effects of GroEL/ES chaperone on the production of soluble form of B. macerans cyclodextrin glucanotransferase (CGTase) in recombinant E. coli were investigated. The cgt gene and groEL/ES genes are under the control of T7 promoter and Pzt-1 promoter, respectively. The optimal concentrations of inducers, IPTG and tetracycline, were found to be 1.0 mM and 10 ng/ml, respectively. When tetracycline and IPTG were added at the early exponential phase (2h) and exponential phase (3h) of growth, respectively, about 1.5-fold increase of soluble CGTase activity and 1.6-fold increase of soluble CGTase protein were obtained. An SDS-PAGE analysis revealed that about $37.2\%$ of total CGTase protein was in the soluble fraction when GroEL/ES chaperone was overexpressed.

• 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.

• 생명과학회지
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• 제14권1호
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• pp.121-125
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• 2004

E. coli에서 B. macerans 유래 cyclodextrin glucanotransferase (CGTase)의 활성형 생산에 GroEL/ES chaperone과의 공발현과 저온 배양의 공동작용 효과에 대해 조사하였다. 실험에 사용된 cgt와 groEL/ES 유전자를 발현하는 pTCGTl과 pGroll은 각각 T7 promoter와 Pzt-1 promoter에 의해 조절되고 이들을 E. coli에 도입시켰다. 대수증식기 초기(2 hr)와 대수증식기 중기(3 hr)에 tetrarycline 10 ng/ml 과 IPTG 1 mM을 첨가하여 각각의 유전자를 발현시켰다. CGTase활성과 specific artivity 측정 시 $37^{\circ}C$에서 pTCGTl 단독 발현 보다 $25^{\circ}C$에서 chaperone과 함께 발현시킨 경우 2배나 높은 활성이 측정됐으며, SDS-PACE 분석결과 $37^{\circ}C$에서 단독 발현 시킬경우 20% 정도 가용성 형태로 발현되던 것이 $25^{\circ}C$에서 chaperone과 공발현 시에는 거의 3.5배가 넘는 69%가 가용성으로 전환됨을 알 수 있었다. 이와 같이 분자 chaperone과 $25^{\circ}C$에서의 저온 배양은 E. coli에서 활성형질 가용성 CGTase의 생산 증가에 큰 영향을 미치는 것으로 나타났다.

• Journal of Microbiology and Biotechnology
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• 제8권5호
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• pp.509-516
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• 1998

A plasmid vector, pXGPRMATG-lac-Tgx, was developed for overproduction of $\beta$-galactosidase in Escherichia coli using the genetic components of groEx, a heat-shock gene cloned from symbiotic X-bacteria in Amoeba proteus. The vector is composed of intragenic promoters P3 and P4 of groEx, the structural gene of lac operon, transcription tenninator signals of lac and groEx, and ColEl and amp'of pBluescript SKII. The optimized host, E. coli DH5$\alpha$, transfonned with the vector constitutively produced 117,310-171,961 Miller units of $\beta$-galactosidase per mg protein in crude extract. The amount of enzyme in crude extract was 53% of total water-soluble proteins. About 43% of the enzyme could be purified to a specific activity of 322,249 Miller units/mg protein after two-fold purification, using two cycles of precipitation with ammonium sulfate and one step of gel filtration. Thus, the expression system developed in this study presents a low-cost and simple method for purifying overproduced $\beta$-galactosidase in E. coli.

• BMB Reports
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• 제38권6호
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• pp.695-702
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• 2005

The groESL bicistronic operon of a restricted facultative methylotrophic bacterium Methylovorus sp. strain SS1 DSM 11726 was cloned and characterized. It was found to consist of two ORFs encoding proteins with molecular masses of 11,395 and 57,396 daltons, which showed a high degree of homology to other bacterial GroES and GroEL proteins. The genes were clustered in the transcription order groES-groEL. Northern blot analyses suggested that the groESL operon is transcribed as a bicistronic 2.2-kb mRNA, the steady-state level of which was markedly increased by temperature elevation. Primer extension analysis demonstrated one potential transcription start site preceding the groESL operon, which is located 100bp upstream of the groES start codon. The transcription start site was preceded by a putative promoter region highly homologous to the consensus sequences of Escherichia coli ${\sigma}^{32}$-type heat shock promoter, which functioned under both normal and heat shock conditions in E. coli. Heat shock mRNA was maximally produced by Methylovorus sp. strain SS1 approximately 10min after increasing the temperature from 30 to $42^{\circ}C$. The groESL operon was also induced by hydrogen peroxide or salt shock.

• Journal of Microbiology and Biotechnology
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• 제15권6호
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• pp.1267-1272
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• 2005

The effects of coexpression of GroEL/ES and DnaK/DnaJ/GrpE chaperones on the productivity of the soluble form of human granulocyte colony stimulating factor (hG-CSF) in E. coli were examined. Recombinant hG-CSF protein was coexpressed with DnaK/DnaJ/GrpE or GroEL/ES chaperones under the control of the araB or Pzt-1 promoter, respectively. The optimal concentration of L-arabinose for the expression of DnaK/DnaJ/GrpE was found to be 1 mg/ml. When L-arabinose was added at $OD_{600}$=0.2 (early-exponential phase), soluble hG-CSF production was greatly increased. In addition, it was observed that the DnaK/DnaJ/GrpE and GroEL/ES chaperones had no synergistic effects on preventing aggregation of hG-CSF protein. Consequently, by coexpression of the DnaK/DnaJ/GrpE chaperone, the signal intensity of the hG-CSF protein band in the soluble fraction of cell lysate was increased from $3.5\%\;to\;13.9\%$, and Western blot analysis also revealed about a 4-5-fold increase of production of soluble hG-CSF over the non-induction case of DnaK/DnaJ/GrpE.