• Journal of Life Science
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• v.12 no.6
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• pp.688-693
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• 2002

Molecular chaperones prevent the misfolding of newly synthesized polypeptides in the cell. The coexpression of molecular chaperones could be expected to improve the production of soluble and active recombinant proteins. In this study, the effect of coexpression of E. coli GroEL/ES chaperone on the active production of Bacillus macerans cyclodextrin glucanotransferase (CGTase) in E. coli was investigated. Two plasmids, pTCGT1 and pGro7 in which the cgt and the groEL/ES genes are under the control of 77 promoter and araB promoter, respectively, were co-transformed into E. coli. With a series of cultures of recombinant E. coli cells, the optimal concentrations of IPTG and L-arabinose were found be 1 mM and 0.3 mg/$m\ell$, respectively. When IPTG and L-arabinose were added at 0.8~1.0 $OD_{600}$ and 0.4~0.5 $OD_{600}$, active CGTase production was increased significantly. This coexpression condition resulted in 1.5-fold increased level of soluble CGTase (0.7~0.73 unit/$m\ell$), compared to the level of CGTase in the single expression (0.36~0.56 unit/$m\ell$). An SDS-PACE analysis revealed that about 33.6% of CGTase in the total CGTase protein was found in the soluble fraction by coexpression of GroEL/ES chaperone.

• Molecules and Cells
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• v.20 no.1
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• pp.142-150
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• 2005

Members of the multifunctional Cyp family have been isolated from a wide range of organisms. However, few functional studies have been performed on the role of these proteins as chaperones in red alga. For studying the function of cDNA GjCyp-1 isolated from the red alga (Griffithsia japonica), we expressed and purified a recombinant GjCyp-1 containing a hexahistidine tag at the amino-terminus in Escherichia coli. An expressed fusion protein, $H_6GjCyp-1$ maintained the stability of E. coli proteins up to $50^{\circ}C$. For a functional bioassay for recombinant $H_6GjCyp-1$, the viability of E. coli cells overexpressing $H_6GjCyp-1$ was compared with that of cells not expressing $H_6GjCyp-1$ at $50^{\circ}C$. After high temperature treatment for 1 h, E. coli overexpressing $H_6GjCyp-1$ survived about three times longer than E. coli lacking $H_6GjCyp-1$. Measurement of the light scattering of luciferase (luc) showed that GjCyp-1 prevents the aggregation of luc during mild heat stress and that the thermoprotective activity of GjCyp-1 is blocked by cyclosporin A (CsA), an inhibitor of Cyps. Furthermore, the Cyp-CsA complex inhibited the growth of E. coli under normal conditions. The results of the GjCyp-1 bioassays as well as in vitro studies strongly suggest that Cyp confers thermotolerance to E. coli.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.4
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• pp.2383-2386
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• 2013

Senescence marker protein 30 (SMP30), a hepatocellular carcinoma (HCC) associated antigen, was earlier shown by our research group to be highly expressed in HCC paracancerous tissues, but have low levels in HCC tissues. In order to detect anti-SMP30 antibody in serum of HCC patients, we established pET30a-SMP30 and pColdIII-SMP30 expression systems in Escherichia coli. However, the expression product was mainly in the form of inclusion bodies. In this research, we used several combinations of chaperones, four molecular chaperone plasmids with pET30a-SMP30 and five molecular chaperone plasmids with pColdIII-SMP30 to increase the amount of soluble protein. Results showed that co-expression of HIS-SMP30 with pTf16, combined with the addition of osmosis-regulator, and a two-step expression resulted in the highest enhancement of solubility. A total of 175 cases of HCC serum were studied by ELISA to detect anti-SMP30 antibody with recombinant SMP30 protein. Some 22 were positive and x2 two-sided tests all showed P>0.05, although it remained unclear whether there was a relationship between positive cases and clinical diagnostic data.

• BMB Reports
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• v.48 no.12
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• pp.685-690
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• 2015

The eukaryotic genome is packed into chromatin, which is important for the genomic integrity and gene regulation. Chromatin structures are maintained through assembly and disassembly of nucleosomes catalyzed by histone chaperones. Asf1 (anti-silencing function 1) is a highly conserved histone chaperone that mediates histone transfer on/off DNA and promotes histone H3 lysine 56 acetylation at globular core domain of histone H3. To elucidate the role of Asf1 in the modulation of chromatin structure, we screened and identified small molecules that inhibit Asf1 and H3K56 acetylation without affecting other histone modifications. These pyrimidine-2,4,6-trione derivative molecules inhibited the nucleosome assembly mediated by Asf1 in vitro, and reduced the H3K56 acetylation in HeLa cells. Furthermore, production of HSV viral particles was reduced by these compounds. As Asf1 is implicated in genome integrity, cell proliferation, and cancer, current Asf1 inhibitor molecules may offer an opportunity for the therapeutic development for treatment of diseases.

• Journal of Microbiology and Biotechnology
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• v.18 no.2
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• pp.299-307
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• 2008

Soybean seed ferritin is essential for human iron supplementation and iron deficiency anemia prevention because it contains abundant bioavailable iron and is frequently consumed in the human diet. However, it is poorly understood in regards its several properties, such as iron mineralization, subunit assembly, and protein folding. To address these issues, we decided to prepare the soybean seed ferritin complex via a recombinant DNA approach. In this paper, we report a rapid and simple Escherichia coli expression system to produce the soybean seed ferritin complex. In this system, two subunits of soybean seed ferritin, H-2 and H-1, were encoded in a single plasmid, and optimal expression was achieved by additionally coexpressing a team of molecular chaperones, trigger factor and GroEL-GroES. The His-tagged ferritin complex was purified by $Ni^{2+}$ affinity chromatography, and an intact ferritin complex was obtained following His-tagged enterokinase (His-EK) digestion. The purified ferritin complex synthesized in E. coli demonstrated some reported features of its native counterpart from soybean seed, including an apparent molecular weight, multimeric assembly, and iron uptake activity. We believe that the strategy described in this paper may be of general utility in producing other recombinant plant ferritins built up from two types of subunits.

• Animal cells and systems
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• v.14 no.1
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• pp.1-10
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• 2010

Previously we showed that CHIP, a co-chaperone of Hsp70 and E3 ubiquitin ligase, can promote the degradation of mutant SOD1 linked to familial amyotrophic lateral sclerosis (fALS) via a mechanism not involving SOD1 ubiquitylation. Here we present evidence that CHIP functions in the interaction of mutant SOD1 with 26S proteasomes. Bag-1, a coupling factor between molecular chaperones and the proteasomes, formed a complex with SOD1 in an hsp70-dependent manner but had no direct effect on the degradation of mutant SOD1. Instead, Bag-1 stimulated interaction between CHIP and the proteasome-associated protein VCP (p97), which do not associate normally. Over-expressed CHIP interfered with the association between mutant SOD1 and VCP. Conversely, the binding of CHIP to mutant SOD1 was inhibited by VCP, implying that the chaperone complex and proteolytic machinery are competing for the common substrates. Finally we observed that mutant SOD1 strongly associated with the 19S complex of proteasomes and CHIP over-expression specifically reduced the interaction between S6/S6' ATPase subunits and mutant SOD1. These results suggest that CHIP, together with ubiquitin-binding proteins such as Bag-1 and VCP, promotes the degradation of mutant SOD1 by facilitating its translocation from ATPase subunits of 19S complex to the 20S core particle.

• BMB Reports
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• v.42 no.7
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• pp.439-443
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• 2009

Dehydrins are group II, late embryogenesis abundant proteins that act putatively as chaperones in stressed plants. To elucidate the function of dehydrins in poplar, we isolated the $SK_2$-type dehydrin gene Podhn from Populus alba $\times$ P. tremula var. glandulosa suspension cells and analyzed its expression following treatments of abiotic stress, wounding and plant growth regulator. Sequence homology and phylogenetic analyses indicate Podhn encodes an acidic dehydrin (pI 5.14, 277 amino acids, predicted size 25.6 kDa) containing two lysine-rich "K-segments" and a 7-serine residue "S-segment", both characteristic of $SK_2$-type dehydrins. Southern blots show Podhn genes form a small gene family in poplar. Podhn was expressed in all tissues examined under unstressed conditions, but most strongly in cell suspensions (especially in the stationary phase). Drought, salt, cold and exogenous abscisic acid (ABA) treatments enhanced Podhn expression, while wounding and jasmonic acid caused its reduction. Therefore, Podhn might be involved in ABA or stress response.

• Journal of Soil and Groundwater Environment
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• v.21 no.5
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• pp.16-24
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• 2016

A new strain of Pseudomonas sp. was isolated from mercury (Hg)-contaminated sites in Taiwan. This bacterium removed more than 80% of Hg present in the culture medium at 12 h incubation and was chosen for further analysis of the molecular mechanisms of Hg tolerance/removal abilities in this Pseudomonas sp. We used RNA-seq, one of the next-generation sequencing methods, to investigate the transcriptomic responses of the Pseudomonas sp. exposed to 60 mg/L of Hg²⁺. We de novo assembled 4,963 contigs, of which 10,533 up-regulated genes and 5,451 down-regulated genes were found to be regulated by Hg. The 40 genes most altered in expression levels were associated with tolerance to Hg stress and metabolism. Functional analysis showed that some Hg-tolerant genes were related to the mer operon, sulfate uptake and assimilation, the enzymatic antioxidant system, the HSP gene family, chaperones, and metal transporters. The transcriptome were analyzed further with Gene Ontology (GO) and Cluster of Orthologous Groups (COGs) of proteins and showed diverse biological functions and metabolic pathways under Hg stress.

• BMB Reports
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• v.48 no.8
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• pp.438-444
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• 2015

Lysosomal storage diseases (LSDs) are a group of inherent diseases characterized by massive accumulation of undigested compounds in lysosomes, which is caused by genetic defects resulting in the deficiency of a lysosomal hydrolase. Currently, enzyme replacement therapy has been successfully used for treatment of 7 LSDs with 10 approved therapeutic enzymes whereas new approaches such as pharmacological chaperones and gene therapy still await evaluation in clinical trials. While therapeutic enzymes for Gaucher disease have N-glycans with terminal mannose residues for targeting to macrophages, the others require N-glycans containing mannose-6-phosphates that are recognized by mannose-6-phosphate receptors on the plasma membrane for cellular uptake and targeting to lysosomes. Due to the fact that efficient lysosomal delivery of therapeutic enzymes is essential for the clearance of accumulated compounds, the suitable glycan structure and its high content are key factors for efficient therapeutic efficacy. Therefore, glycan remodeling strategies to improve lysosomal targeting and tissue distribution have been highlighted. This review describes the glycan structures that are important for lysosomal targeting and provides information on recent glyco-engineering technologies for the development of therapeutic enzymes with improved efficacy. [BMB Reports 2015; 48(8): 438-444]

• Toxicological Research
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• v.24 no.2
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• pp.129-135
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• 2008

The results of recent studies indicate that high levels of free fatty acids(FFAs) and adipokines may be the main causes of non-alcoholic liver disease; however, the molecular mechanism that links FFAs to lipotoxicity remains unclear. In the present study, we treated HepG2 cells with FFA(either palmitate or oleate) to investigate the mechanisms involved in lipotoxicity in the liver cells. We also treated cells with palmitate in the presence of a chemical chaperone, 4-phenylbutyric acid(PBA), to confirm the involvement of ER stress in lipotoxicity. Palmitate significantly induced cytotoxicity in dose- and time-dependent manners. Apoptosis was also significantly induced by palmitate as measured by caspase-3 activity and DAPI staining. Palmitate led to increased expressions of the spliced form of X-box-protein(Xbp)-1 mRNA and C/EBP homologous transcription factor(CHOP) protein, suggesting activation of the unfolded-protein response. PBA co-incubation significantly attenuated apoptosis induced by palmitate. The above data demonstrate that high levels of palmitate induce apoptosis via the mediation of ER stress in the liver cells and that chemical chaperones act to modulate ER stress and accompanying apoptosis.