• BMB Reports
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• v.41 no.2
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• pp.108-111
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• 2008

Some proteins of E. coli are stable at temperatures significantly higher than $49^{\circ}C$, the maximum temperature at which the organism can grow. The heat stability of such proteins would be a property which is inherent to their structures, or it might be acquired by evolution for their specialized functions. In this study, we describe the identification of 17 heat-stable proteins from E. coli. Approximately one-third of these proteins were recognized as having functions in the protection of other proteins against denaturation. These included chaperonin (GroEL and GroES), molecular chaperones (DnaK and FkpA) and peptidyl prolyl isomerases (trigger factor and FkpA). Another common feature was that five of these proteins (GroEL, GroES, Ahpc, RibH and ferritin) have been shown to form a macromolecular structure. These results indicated that the heat stability of certain proteins may have evolved for their specialized functions, allowing them to cope with harsh environments, including high temperatures.

• 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.

• Animal cells and systems
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• v.6 no.3
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• pp.227-232
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• 2002

GroESLx proteins of symbiotic X-bacteria were overproduced in Escherichia coli and their structural characteristics were assayed after simple purification. The GroESx and GroELx were heat-stable at 8$0^{\circ}C$ and 5$0^{\circ}C$, respectively. After heat-treatment, GroESx was purified by DEAE Sephadex A-50 chromatography and GroELx was purified by step- and linear sucrose density gradient ultracentrifugation. Molecular masses of GroESx and GroELx were 50-80 kDa and 800 kDa, respectively, as estimated by sucrose density gradient ultracentrifugation. In chemical cross-linking analysis, subunits of GroESx were mostly cross-linked by incubation for 3 h in 0.4% glutaralde-hyde and GroESx was found to be composed of homo-heptamer subunits. Those of GroELx were cross-linked within 10 min in 0.3% glutaraldehyde and GroELx was in two stacks of homo-heptamer subunits. On the other hand, GroESx and GroELx proteins in a solution could not be cross-linked even after incubation for 3 h in 0.5% glutaraldehyde. GroELx was stable at 4-37$^{\circ}C$. In the presence of both GroESx and ATP, GroELx$_{14}$ was stable at 37$^{\circ}C$ but not at 4$^{\circ}C$ or 24$^{\circ}C$. Thus, we confirmed the oligomeric properties of GroESx$_{7}$ and GroELx$_{14}$ and their stability to heat and in the interaction with GroESx.x.

• 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.

• BMB Reports
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• v.40 no.6
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• pp.936-943
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• 2007

Previously it was reported that promoter of groES-groEL operon of Staphylococcus aureus is induced by various cellwall active antibiotics. In order to exploit the above promoter for identifying novel antistaphylococcal drugs, we have cloned the promoter containing region ($P_g$) of groES-groEL operon of S. aureus Newman and found that the above promoter is induced by sublethal concentrations of many antibiotics including cell-wall active antibiotics. A reporter S. aureus RN4220 strain (designated SAU006) was constructed by inserting the $P_g$-lacZ transcriptional fusion into its chromosome. Agarose-based assay developed with SAU006 shows that $P_g$ in single-copy is also induced distinctly by different classes of antibiotics. Data indicate that ciprofloxacin, rifampicin, ampicillin, and cephalothin are strong inducers, whereas, tetracycline, streptomycin and vancomycin induce the above promoter weakly. Sublethal concentrations of ciprofloxacin and ampicilin even have induced $P_g$ efficiently in microtiter plate grown SAU006. Additional studies show for the first time that above promoter is also induced weakly by arsenate salt and hydrogen peroxide. Taken together, we suggest that our simple and sensitive assay systems with SAU006 could be utilized for screening and detecting not only novel antistaphylococcal compounds but also different toxic chemicals.

• KSBB Journal
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• v.21 no.2
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• pp.118-122
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• 2006

In this report, we describe that the use of GroEL/GroES-enriched S30 extract remarkably enhances the solubility and enzymatic activity of cell-free synthesized rPA, which requires the correct formation of 9 disulfide bonds for its biological activity. We found that the stable maintenance of redox potential is necessary, but not sufficient for the optimal expression of active rPA. In a control reaction without using additional molecular chaperones, most of the rPA molecules were aggregated almost instantly after their expression and thus failed to exhibit the enzymatic activity. However, by the use of GroEL/GroES-enriched extract, combined with IAM-treatment, approximately $30{\mu}g/ml$ of active rPA was expressed in the cell-free synthesis reaction. This result not only demonstrates the efficient production of complex proteins, but also shows the control and flexibility offered by the cell-free protein synthesis system.

• Journal of the Korean Magnetic Resonance Society
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• v.17 no.1
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• pp.30-39
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• 2013

Immortalization-upregulated protein-1 (IMUP-1) genes have been cloned and are known to be involved in SV40-mediated immortalization. IMUP-1 gene is highly expressed in various cancer cell lines and tumors, suggesting the possibility that they might be involved in tumorigenicity. Previously, there were several problems for overexpression of IMUP-1 in bacterial expression systems including low solubility and aggregation due to unstructured property. To investigate the structural properties, it is necessary to obtain lots of pure and soluble proteins. Accordingly, the co-expression systems of bacterial chaperone proteins, GroEL-GroES, were used to increase solubility of IMUP-1. From the analysis of NMR and CD experiment data, it is suggested that the protein adopt typical the random coil properties in solution.

• 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.

• Journal of Microbiology and Biotechnology
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• v.30 no.2
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• pp.259-270
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• 2020

Listeria monocytogenes is a gram-positive, facultative anaerobe food pathogen responsible for the listeriosis that mostly occurs during the low-temperature storage of a cold cut or dairy products. To understand the systemic response to a wide range of growth temperatures, L. monocytogenes were cultivated at a different temperature from 10℃ to 42℃, then whole cell proteomic analysis has been performed both exponential and stationary cells. The specific growth rate increased proportionally with the increase in growth temperature. The maximum growth rate was observed at 37℃ and was maintained at 42℃. Global protein expression profiles mainly depended on the growth temperatures showing similar clusters between exponential and stationary phases. Expressed proteins were categorized by their belonging metabolic systems and then, evaluated the change of expression level in regard to the growth temperature and stages. DnaK, GroEL, GroES, GrpE, and CspB, which were the heat&cold shock response proteins, increased their expression with increasing the growth temperatures. In particular, GroES and CspB were expressed more than 100-fold than at low temperatures during the exponential phase. Meanwhile, CspL, another cold shock protein, overexpressed at a low temperature then exponentially decreased its expression to 65-folds. Chemotaxis protein CheV and flagella proteins were highly expressed at low temperatures and stationary phases. Housekeeping proteins maintained their expression levels constant regardless of growth temperature or growth phases. Most of the growth related proteins, which include central carbon catabolic enzymes, were highly expressed at 30℃ then decreased sharply at high growth temperatures.

• 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.