• Journal of Microbiology
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• v.34 no.1
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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.

• Korean Journal of Veterinary Research
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• v.45 no.1
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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.

• Journal of Microbiology
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• v.38 no.3
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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$^{\circ}C$.

• Korean Journal of Microbiology
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• v.32 no.1
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• pp.47-52
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• 1994

The cells of Campylobacter jejuni heat-shocked at 48${\circ}C$ for 30 min synthesized the heat shock proteins of HSP90, HSP66 and HSP60. Those heat shock proteins were found to correspond to the heat shock proteins of HSP87, HSP66 (DnaK), and HSP58 (GroEL) of E. coli, respectively. By Southern blot analysis of the chromosomal DNAs of C. jejuni with groESL and dnaK genes of E. coli as DNA probes, the heat shock genes of C. jejuni which are homologous to the E. coli groESL and dnaK genes were found to exist in the chromosomal DNA. The genomic libraries of C. jejuni were constructed with the cosmid vector pWE15 and the groEL gene of C. jejuni were cloned in E. coli B178 groEL44 temperature senstive mutant. The hybrid plasmid (pLC1) was inserted with the DNA fragment (about 5.7kb in size) containing the groEL gene. E. coli groEL44 mutant cell transformed with the pLC1 could grow at 42${\circ}C$ by synthesizing the HSP60 of C. jejuni and regained the susceptibility to the ${\lambda}$ vir phage by expression of the groEL gene in the cloned cells. These indicated that the groEL products of C. jejuni had chaperon effects by synthesizing the heat shock proteins in the cloned cells of E. coli.

• Journal of Microbiology and Biotechnology
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• v.26 no.2
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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.

• Korean Journal of Microbiology
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• v.30 no.6
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• pp.421-424
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• 1992

Escherichia coli JM83 harboring penicilin G acylase gene of Bacillus megaterium ATCC14945 produced a protein in large amount (>20% of the total protein). The protein was identified as GroEL, one of the E. coli heat shock protein, by N-terminal amino acid sequence analysis. It was found that GroEL was induced by the expressed foreign penicilin G acylase at both 27 and $37^{\circ}C$.

• KSBB Journal
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• v.23 no.5
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• pp.403-407
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• 2008

Recently, Candida antarctica lipase B (CalB) draws attention from industries for various applications for food, detergent, fine chemical, and biodiesel, because of its characteristics as an efficient biocatalyst. Since many industrial processes carry out in organic solvent and at high temperature, CalB, which is stable under harsh condition, is in demand from many industries. In order to reform CalB promptly, the expression system which has advantages of ease to use and low cost for gene libraries screening was developed using E. coli. The E. coli strains, Rosettagami with competence for enhanced disulfide bond formation, Novablue, and $DH5{\alpha}$, were exploited in this study. To obtain the soluble CalB, the pCold I vector expressing the cloned gene at $15^{\circ}C$ and the chaperone plasmids containing groES/groEL, groES/groEL/tig, tig, dnaK/dnaJ/grpE, and dnaK/dnaJ/grpE/groES/groEL were used for coexpression of CalB and chaperones. The colonies expressing functional lipase were selected by employing the halo plate containing 1% tributyrin, and the CalB expression was confirmed by SDS-PAGE. E. coli Rosettagami and $DH5{\alpha}$ harbouring groES/groEL chaperones were able to express soluble CalB effectively. From a facilitative point of view, E. coli $DH5{\alpha}$ is more suitable for further mutation study.

• Journal of Microbiology and Biotechnology
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• v.8 no.5
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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.

• Journal of Microbiology and Biotechnology
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• v.12 no.6
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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.

• Korean Journal of Microbiology
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• v.38 no.3
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• pp.162-167
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• 2002

To analyse proteins and gene related to antifungal activity, SAR01 strain was isolated from a brown seaweed and identified as Streptomyces sp. by FAME(fatty acid methyl ester) analysis. Antifungal activity deficient mutant(SAR535) of Streptomyces sp. SAR01 was induced by gamma radiation$({60}^Co)$. It was found that 6 specific protein spots appeared only in SAR01 by 2-D electrophoresis analysis. Among them, a protein of 10 kDa had homology of 96% with 10 kD chaperonin cpn 10 (GroES) by Basic Local Alignment Search Tool(BLAST, NCBI) analysis. SAR535 transformants into which groES was transferred by electroporation revealed antifungal activity newly similar with SAR01 It suggested that groES be supposed to be related to the antifungal activity of Streptomyces sp. SAR01.