• 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 Life Science
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• v.14 no.1
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• pp.121-125
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• 2004

The synergistic effect of lowered incubation temperature and CroEL/ES expression on the production of soluble form of B. macerans cyclodextrin glucanotransferase (CGTase) was studied in recombinant E. coli. pTCGTl and pGroll carrying the cgt and groEL/ES genes under the control of T7 promoter and pzt-I promoter, respectively, were co-introduced. Tetracycline (10 ng/ml) and IPTG (1 mM) were added at the early-exponential phase (2 hr) and mid-exponential phase (3 hr). Low temperature cultivation at $25^{\circ}C$ with groEL/ES expression improved the activity of CGTase by two fold, compared to $37^{\circ}C$ cultivation without chaperones. SDS-PACE analysis revealed that about 69% of CGTase in the total CGTase protein was found in the soluble fraction by overexpression of GroEL/ES and cultivation at$25^{\circ}C$, whereas 20% of CGTase was detected in the soluble fraction when E. coli was cultivated at $37^{\circ}C$ without chaperone. The amount of soluble CGTase from $25^{\circ}C$ culture with chaperone was 3.5-fold higher than that of $37^{\circ}C$ culture without chaperone. Therefore the expression of CroEL/ES and low temperature cultivation greatly enhanced the soluble production of CGTase in E. coli.

• Journal of Microbiology and Biotechnology
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• v.28 no.5
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• pp.739-747
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• 2018

The low solubility and high-cost recovery of Propionibacterium acnes polyunsaturated fatty acid isomerase (PAI) are key problems in the bioproduction of high value-added conjugated linoleic acid (CLA). To improve the solubility of recombinant PAI, six chaperone proteins were coexpressed with PAI. Introduction of GroELS proteins dramatically improved the PAI solubility from 29% to 97%, with increased activity by 57.8%. Combined expression of DnaKJ-GrpE and GroELS proteins increased the activity by 11.9%. In contrast, coexpression of DnaKJ-GrpE proteins significantly reduced the activity by 57.4%. Plasmids pTf16 harboring the tig gene and pG-Tf2 containing the tig and groEL-groES genes had no visible impact on PAI expression. The lytic protein E was then introduced into the recombinant Escherichia coli to develop a cell autolysis system. A 35% activity of total intracellular PAI was released from the cytoplasm by suspending the lysed cells in distilled water. The PAI recovery was further improved to 81% by optimizing the release conditions. The lipase from Rhizopus oryzae was also expressed in E. coli, with an extracellular activity of 110.9 U/ml. By using the free PAI and lipase as catalysts, a joint system was established for producing CLA from sunflower oil. Under the optimized conditions, the maximum titer of t-10,c-12-CLA reached 9.4 g/l. This work provides an effective and low-cost strategy to improve the solubility and recovery of the recombinant intracellular PAI for further large-scale production of CLA.

• BMB Reports
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• v.33 no.1
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• pp.75-81
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• 2000

DnaK is a major heat shock protein and known to be highly conserved in all species. Previously, the dnaK in Streptococcus pneumoniae was cloned and the immunogenic nature characterized. In this study, dnaK mutants were generated by insertion of duplication mutagenesis and their characteristics examined. They had defective growths at all temperatures ($20^{\circ}C-42^{\circ}C$)and cell divisions, and formed filaments after a temperature shift from 30 to 42. A unique feature of the dnaK mutants of S. pneumoniae, unlike those of E. coli and B. subtilis, was the growth capability at high temperature ($42^{\circ}C$) without producing the putative GroEL. Our results suggest that DnaK may serve as a regulator and/or modifier in GroEL gene expression.

• 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.

• Fisheries and Aquatic Sciences
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• v.16 no.4
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• pp.273-277
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• 2013

Vibriosis caused by Vibrio anguillarum and edwardsiellosis caused by Edwardsiella tarda are septicemic diseases of many commercially important freshwater and marine fishes, and threaten the aquaculture industry in Korea. Early diagnosis and accurate identification of these two bacterial species could help to prevent these diseases and minimize the damage to cultured marine species. This study designed a duplex polymerase chain reaction (PCR) method for the simultaneous detection of two major fish pathogens: V. anguillarum and E. tarda. Each pair of oligonucleotide primers exclusively amplified the target groEL gene of the specific microorganism. Twenty-two Vibrio and ten non-Vibrio enteric species were used to check the specificity of the primers, which were found to be highly specific for the target species, even among closely related species. The detection limit was 400 pg for V. anguillarum and 4 ng for E. tarda when mixed purified DNA was used as the template. This assay showed high specificity and sensitivity in the simultaneous detection of V. anguillarum and E. tarda from artificially inoculated seawater and fish.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 2001.06a
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• pp.62-72
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• 2001

The N-carbamoyl-D-amino acid amidohydrolase (D-carbamoylase) gene (dcb) from Agrobacterium tumefaciens AM 10 has been successfully cloned and expressed in Escherichia coli. Expression of D-carbamoylase gene under the 17 promoter in different host strains showed that the optimal expression was achieved in E. coli JM109 (DE3) with a 9-fold increase in enzyme production compared to the wild-type strain. The co-expression of the GroEL/ES protein with D-carbamoylase protein caused an in vivo solubilization of D-carbamoylase in an active form. The synergistic effect of GroEL/ES at 28$^{\circ}C$ led to 60 ％ solubilization of the total expressed target protein with a 6.2-fold increase in enzyme activity in comparison to that expressed without GroEL/ES and 43-fold increase in enzyme activity compared to A. tumefaciens AM 10. Attempts to express D-carbamoylase in an altered redox cytoplasmic milieu did not improve the enzyme production in an active form. The Histidyl-tagged D-carbamoylase was purified in a single step by Nickel-affinity chromatography and was found to have a specific activity of 9.5 U/mg protein.

• Korean Journal of Microbiology
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• v.37 no.3
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• pp.221-227
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• 2001

Heat shock proteins serve as chaperone by preventing the aggregation of denatured proteins and promote survival of pathogens in harsh environments. In bacteria, ethanol shock induced the major chaperone GroEL and DnaK, but in Streptococcus pneumoniae, it induced neither GroEL nor DnaK but alcohol dehydrogenase (ADH). In this study, ADH gene encoding a 104-kDa (p104) protein was identified and characterized. The deduced amino acid sequence of pneumococcal ADH shows homology with other members of the ADH family, and particularly with Entamoeba histolytica ADH2 and E. coli ADH. S. pneumoniae adh is composed of 883 amino acids and its estimated isoelectric point is 6.09. Although ADH is conserved between S. pneumoniae and E. coli, immunoblot analysis employing antisera raised against pneumococcus ADH demonstrated no cross-reactivity with ADH analog in Eschericha coli, Staphylococcus aureus and human HeLa cells. Also secretion of ADH was demonstrated by subcellular fractionation and immunoblot analysis of proteins. These results suggest that S. pneumoniae ADH could be a highly feasible candidate for both diagnostic marker and vaccine.

• Journal of Life Science
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• v.6 no.3
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• pp.213-218
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• 1996

Symbionin, ahomologue of E. coli GroEL, produced by an intracellular symbiont of the pea aphid , has molecular chaperone activity bothin vitro and in vivo, and it is able to tarnsfer its high-energy phospholy group to other compounds through its autophosphorylation and phosphotransferase activity. The symbionin is a novel histidine protein Kinase and a senor molecular of the two-component pathway.

• Proceedings of the Zoological Society Korea Conference
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• 1996.10a
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• pp.215.2-215
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• 1996

No Abstract, See Full Text