• Molecules and Cells
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• v.24 no.2
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• pp.210-214
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• 2007

The 90-kDa heat shock protein (HSP90) normally functions as a molecular chaperone participating in folding and stabilizing newly synthesized proteins, and refolding denatured proteins. The HSP90 inhibitor geldanamycin (GA) occupies the ATP/ADP binding pocket of HSP90 so inhibits its chaperone activity and causes subsequent degradation of HSP90 client proteins by proteasomes. Here we show that GA reduces the level of endogenous c-Jun in human embryonic kidney 293 (HEK293) cells in a time and dose dependent manner, and that this decrease can be reversed by transfection of HSP90 plasmids. Transfection of HSP90 plasmids in the absence of GA increases the level of endogenous c-Jun protein, but has no obvious affect on c-Jun mRNA levels. We also showed that HSP90 prolongs the half-life of c-Jun by stabilizing the protein; the proteasome inhibitor N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG132) blocks the degradation of c-Jun promoted by GA. Transfection of HSP90 plasmids did not obviously alter phosphorylation of c-Jun, and a Jun-2 luciferase activity assay indicated that over-expression of HSP90 elevated the total protein activity of c-Jun in HEK293 cells. All our evidence indicates that HSP90 stabilizes c-Jun protein, and so increases the total activity of c-Jun in HEK293 cells.

• Journal of Life Science
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• v.29 no.4
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• pp.492-498
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• 2019

Previously, three kinds of lipases, lipAD1, lipAD2, and lipAD3, and lipase chaperone, lipBD, of Acinetobacter schindleri DYL129 isolated from soil sample were reported. In this report, three lipase and lipase chaperone were cloned into the pET32a(+) or pGEX-6P-1 vectors for protein expression in Escherichia coli, and named as pETLAD1, pETLAD2, pETLAD3 and pETLBD or pGEXLAD1, pGEXLAD 2, pGEXLAD3 and pGEXLBD, respectively. Protein expression rate was higher in pET system than in pGEX system. Although LipAD1 and LipAD2 were produced as inclusion bodies, their expression levels were high. So LipAD1 and LipAD2 could be solubilized in 8 M urea buffer and purified. LipAD3 and LipBD were overexpressed in soluble form and purified. Those proteins were purified by His-tag affinity chromatography connected in AKTA prime system. The activities of the purified lipases were demonstrated with 1% tributyrin agar plate. After purification, molecular mass was determined with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. LipAD1 showed high activity toward ${\rho}$-nitrophenyl acetate and ${\rho}$-nitrophenyl butyrate, LipAD2 showed high activity toward ${\rho}$-nitrophenyl acetate and ${\rho}$-nitrophenyl myristate, and LipAD3 showed high activity toward ${\rho}$-nitrophenyl acetate, ${\rho}$-nitrophenyl butyrate, and ${\rho}$-nitrophenyl miristate, respectively. Three lipases, LipAD1, LipAD2, and LipAD3, showed optimal reaction at $50^{\circ}C$ using ${\rho}$-nitrophenyl butyrate, as substrate.

• International Journal of Industrial Entomology and Biomaterials
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• v.7 no.2
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• pp.127-131
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• 2003

Protein disulfide isomerase (PDI) found in the endoplasmic reticulum (ER) catalyzes disulfide bond exchange and assists in protein folding of newly synthesized proteins. PDI also functions as a molecular chaperone and has been found to be associated with proteins in the ER. In addition, PDI functions as a subunit of two more complex enzyme systems: the prolyl-4-hydroxylase and the triacylglycerol transfer proteins. A cDNA that encodes protein disulfide isomerase was previously isolated from Bombyx mori (bPDI), in which open reading frame of 494 amino acids contained two PDI-typical thioredoxin active site of WCGHCK and an ER retention signal of the KDEL motif at its C-terminal, and we report its functional characterization here. This putative bPDI cDNA is expressed in insect Sf9 cells as a recombinant proteins using baculovirus expression vector system. The bPDI recombinant proteins are successfully recognized by antirat PDI antibody, and shown to be biologically active in vitro by mediating the oxidative refolding of reduced and scrambled RNase. This suggests that bPDI may play an important role in protein folding mechanism of insects.