• Proceedings of the Korean Society of Sericultural Science Conference
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• 2001.10a
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• pp.30-32
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• 2001

no Abstract, See Full Text

• Biotechnology and Bioprocess Engineering:BBE
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• v.11 no.5
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• pp.414-419
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• 2006

When the alginate lyase gene (aly) from Pseudoalteromonas elyakovii was expressed in E. coli, most of the gene product was organized as aggregated insoluble particles known as inclusion bodies. To examine the effects of chaperones on soluble and nonaggregated form of alginate lyase in E. coli, we constructed plasm ids designed to permit the coexpression of aly and the DnaK/DnaJ/GrpE or GroEL/ES chaperones. The results indicate that coexpression of aly with the DnaK/DnaJ/GrpE chaperone together had a marked effect on the yield alginate lyase as a soluble and active form of the enzyme. It is speculated this result occurs through facilitation of the correct folding of the protein. The optimal concentration of L-arabinose required for the induction of the DnaK/DnaJ/GrpE chaperone was found to be 0.05mg/mL. An analysis of the protein bands on SDS-PAGE gel indicated that at least 37% of total alginate lyase was produced in the soluble fraction when the DnaK/DnaJ/GrpE chaperone was coexpressed.

• Journal of Microbiology and Biotechnology
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• v.15 no.6
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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.

• 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.

• BMB Reports
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• v.42 no.10
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• pp.623-630
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• 2009

Hsp90, an evolutionarily conserved molecular chaperone, is involved in the folding, stabilization, activation, and assembly of a wide range of 'client' proteins, thus playing a central role in many biological processes. Especially, several oncoproteins act as Hsp90 client proteins and tumor cells require higher Hsp90 activity than normal cells to maintain their malignancy. For this reason, Hsp90 has emerged as a promising target for anti-cancer drug development. It is still largely unknown how Hsp90 can recognize structurally unrelated client proteins. However, recent progress in structural studies on Hsp90 and its interaction with various co-chaperones has broadened our knowledge of how the Hsp90 ATPase activity, which is essential for its chaperone function, is regulated and coupled with the conformational changes of Hsp90 dimer. This review focuses on the roles of various Hsp90 co-chaperones in the regulation of the Hsp90 ATPase cycle, as well as in the selection of client proteins. In addition, the current development of Hsp90 inhibitors based on the structural information will be discussed.

• Journal of Life Science
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• v.9 no.1
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• pp.76-83
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• 1999

Mammalial expression vectors containing GRP94, BiP, ERp72, and PDI, were introduced into FRTL5 cells. Transfected cells were selected by neomycin resistance for exogenously overexpressed proteins in the ER. The use of a reducible cross-linker, DSP, markedly improved the ability to detect noncovalent interactions of PDI, BiP and GRP94 with newly-synthesized thyroglobulin. Under normal conditions, GRP94 was found to associate transiently with early Tg folding intermediates, displaying interaction kinetics similar to those reported for another ER chaperones of BiP.

• Molecules and Cells
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• v.19 no.3
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• pp.328-333
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• 2005

Small heat shock proteins (sHSPs) are widely distributed, and their function and diversity of structure have been much studied in the field of molecular chaperones. In plants, which frequently have to cope with hostile environments, sHSPs are much more abundant and diverse than in other forms of life. In response to high temperature stress, sHSPs of more than twenty kinds can make up more than 1% of soluble plant proteins. We isolated a genomic clone, NtHSP18.3, from Nicotiana tabacum that encodes the complete open reading frame of a cytosolic class I small heat shock protein. To investigate the function of NtHSP18.3 in vitro, it was overproduced in Escherichia coli and purified. The purified NtHSP18.3 had typical molecular chaperone activity as it protected citrate synthase and luciferase from high temperature-induced aggregation. When E. coli celluar proteins were incubated with NtHSP18.3, a large proportion of the proteins remained soluble at temperatures as high as $70^{\circ}C$. Native gel analysis suggested that NtHSP18.3 is a dodecameric oligomer as the form present and showing molecular chaperone activity at the condition tested. Binding of bis-ANS to the oligomers of NtHSP18.3 indicated that exposure of their hydrophobic surfaces increased as the temperature was raised. Taken together, our data suggested that NtHSP18.3 is a molecular chaperone that functions as a dodecameric complex and possibly in a temperature-induced manner.

• Journal of Physiology & Pathology in Korean Medicine
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• v.16 no.4
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• pp.680-683
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• 2002

Bovine aortic endothelial cells(BAEC) produce two variant forms of laminin with a subunit composition of AB1B2 and A'B1B2. Analyses of the intracellular assembly of these subunits revealed that the B1B2 dimer formed first, and that A or A' joined to form the AB1B2 or A'B1B2 trimer. Angiostatic steroids shifted the relative size of the A and A' monomer pool in BAEC, and competition between the A and A' subunits in joining the B1B2 dimer produced AB1B2 and A'B1B2 in different ratios. This result suggests that subunit replacement is the general mechanism for producing laminin variants by various cells for tissue morphogenesis. When laminin subunits in BAEC were cross-linked with dithio-bis-succinimidylpropionate(DSP) and immunoprecipitated with anti-Iaminin antiserum, monomeric A,A',B1 and B2 monomers and the B1B2 dimer migrated as extremely large molecules in sodium dodecyl sulfate gel electrophoresis under nonreducing conditions. When the crosslinking disulfide bonds were cleaved under reducing conditions, they migrated as the usual subunits. This result suggests that molecular chaperones were involved in the process of the assembly and replacement of laminin subunits.

• Molecules and Cells
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• v.21 no.3
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• pp.381-388
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• 2006

Heat shock proteins (Hsp) 70 are a ubiquitous family of molecular chaperones involved in many cellular processes. A yeast strain, ssa1/2, with two functionally redundant cytosolic Hsp70s (SSA1 and SSA2) deleted shows thermotolerance comparable to mildly heatshocked wild type yeast, as well as increased protein synthesis and ubiquitin-proteasome protein degradation. Since mRNA abundance does not always correlate well with protein expression levels it is essential to study proteins directly. We used a gel-based approach to identify stress-responsive proteins in the ssa1/2 mutant and identified 43 differentially expressed spots. These were trypsin-digested and analyzed by nano electrospray ionization liquid chromatography tandem mass spectrometry (nESI-LC-MS/MS). A total of 22 non-redundant proteins were identified, 11 of which were confirmed by N-terminal sequencing. Nine proteins, most of which were up-regulated (2-fold or more) in the ssa1/2 mutant, proved to be stress-inducible proteins such as molecular chaperones and anti-oxidant proteins, or proteins related to carbohydrate metabolism. Interestingly, a translational factor Hyp2p up-regulated in the mutant was also found to be highly phosphorylated. These results indicate that the cytosolic Hsp70s, Ssa1p and Ssa2p, regulate an abundance of proteins mainly involved in stress responses and protein synthesis.

• Microbiology and Biotechnology Letters
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• v.43 no.3
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• pp.212-218
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• 2015

Fucosyltransferases (FucTs) catalyze fucosyl transfer from guanosine-diphosphate fucose (GDP-β-L-fucose) to acceptor molecules to form fucosyloligosaccharides with α-glycosidic linkages. However, when FucT genes have been expressed in Escherichia coli, most cases have resulted in the production of inclusion bodies. In this study, to overcome this drawback, molecular chaperones were co-expressed with α1,2-fucosyltransferase (FucT2) in E. coli. For this, the pACYC184 vector, having genes for chaperones such as GroEL, GroES, DnaK, DnaJ, and GrpE, were transformed into E. coli BL21 (DE3) star harboring pHFucT2, including the FucT2 gene from Helicobacter pylori 26695. The results from SDS-PAGE showed that 5 chaperones were successfully expressed and the soluble fraction of FucT2 was also increased. HPLC analysis revealed that the coexpression of chaperone proteins resulted in a 5-fold increase in the total activity of fucosyltransferase in E. coli. In conclusion, the FucT2 expression system developed in this study can be used as a useful tool for the synthesis of fucosyloligosaccharides.