• BMB Reports
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• v.38 no.6
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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.

• Korean Journal of Microbiology
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• v.30 no.3
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• pp.232-238
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• 1992

Canz~~j~lohuc;tc.~jurn i werc studied for their heat shock responses at several elevated temperatures and their heat shock genes were detected by the technique of Southern hybridization. (.. ,jc\ulcorneruni sy~>thesized the major heat shock proteins of hsp90. hsphh. and hsphO at 48$^{\circ}$C . ant1 their w~u.ival rates were maintained as the same level at optimal temperature. '1-hc heat shock genes in chromosome of C ,jc:jutii werc determined to be homologous to the heat shock genes or E. t,oli. by showing strong signals in Southern hybridization analysis using clnaK and groESL- as DNA probe But the restriction sites for thc fragmcnts including heat shock genes were different betueen E. c,oli and C ,jtjuni.

• 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.

• Proceedings of the PSK Conference
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• 2001.04a
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• pp.180.2-180.2
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• 2001

• Biotechnology and Bioprocess Engineering:BBE
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• v.3 no.2
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• pp.67-70
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• 1998

In order to understand the influence of ferroxidase center on the protein assembly and solubility of tadpole ferrin, three mutant plasmids, pTH58K, pTH61G, and pTHKG were constructed with the aid of site-directed mutagenesis and mutant proteins were produced in Eshcerichia coli. Mutant ferritin H-subunits produced by the cells carrying plasmids pTH58K and pTHKG were active soluble proteins, whereas the mutant obtained from the plasmid pTH61G was soluble only under osmotic stress in the presence obtained from the plasmid pTH61G was soluble only under osmotic stress in the presence of sorbitol and betaine. Especially, the cells carrying pTH61G together with the plasmid pGroESL harboring the molecular chaperone genes produced soluble ferritin. The mutant ferritin H-subunits were all assembled into ferritin-like holoproteins. These mutant ferritns were capable of forming stable iron cores, which means the mutants are able to accumulate iron with such modified ferroxidase sites. Further functional analysis was also made on the individual amino acid residues of ferroxidase center.

• Korean Journal of Microbiology
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• v.37 no.4
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• pp.245-252
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• 2001

Erm proteins, MLS (macrolide-lincosamide-streptogramin B) resistance factor proteins, show high degree of amino acid sequence homology and comprise of a group of structurally homologous N-methyltransferases. On the basis of the recently determined structures of ErmC` and ErmAM, ErmSF was divided into two domains, N-terminal end catalytic domain and C-terminal end substrate binding domain and attempted to overexpress catalytic domain in E. coli using various pET expression systems. Three DNA fragments were used to express the catalytic domain: DNA fragment 1 encoding Met 1 through Glu 186, DNA fragment 2 encoding Arg 60 to Glu 186 and DNA fragment 3 encoding Arg 60 through Arg 240. Among the pET expression vectors used, pET 19b successfully expressed the DNA fragment 3 and pET23b succeeded in expression of DNA fragment 1 and 2. But the overexpressed catalytic domains existed as inclusion body, a insoluble aggregate. To assist the soluble expression of ErmSF catalytic domains, Coexpression of chaperone GroESL or Thioredoxin and lowering the incubation temperature to $22^{\circ}C$ were attempted, as did in the soluble expression of the whole ErmSF protein. Both strategies did not seem to be helpful. Solubilization with guanidine-HCl and renaturation with gradual removal of denaturant and partial digestion of overexpressed whole ErmSF protein (expressed to the level of 126 mg/ι culture as a soluble protein) with proteinase K, nonspecific proteinase are under way.

• Korean Journal of Microbiology
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• v.43 no.3
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• pp.166-172
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• 2007

ERM proteins transfer the methyl group to $A_{2058}$ in 23S rRNA, which reduces the affinity of MLS (macrolide-lincosamide-streptogramin B) antibiotics to 23S rRNA, thereby confer the antibiotic resistance on micro-organisms ranging from antibiotic producers to pathogens and are classified into monomethyltransferase and dimethyltransferase. To investigate the differences between mono- and dimethyltransferase, tirD, a representative monomethylase gene was cloned in Escherichia coli from Streptomyces fradiae which contains ermSF, dimethylase gene as well to overexpress the TlrD for the first time. T7 promoter driven expression system successfully overexpress tlrD as a insoluble aggregate at $37^{\circ}C$ accumulating to around 55% of the total cell protein but unlike ErmSF, culturing at temperature as low as $18^{\circ}C$ did not make insoluble aggregate of protein into soluble protein. Coexpression of Thioredoxin and GroESL, chaperone was not helpful in turning into soluble protein either as in case of ErmSF. These results might suggest that differences between mono- and dimethylase could be investigated on the basis of the characteristics of protein structure. However, a very small amount of soluble protein which could not be detected by SDS-PAGE conferred antibiotic resistance on E. coli as in ErmSF which was expected from the activity exerted by monmethylase in a cell.