ABSTRACT
Proteasome is a multi-subunit protease complex in eukaryocytes, and plays an important role in ubiquitin-proteosome pathway. Recombinant proteasome can be used to screen proteasome inhibitors. In this study, recombinant plasmid of pET28a-PSMB1 was constructed by inserting human proteasome catalytic subunit (PSMB1) cDNA (726 bp) into the prokaryotic expression vector pET28a(+), and transforming the plasmid into E. coli BL21(DE3) cells for expression. After overnight induction (1 mmol/L IPTG, 20 degrees C), an expected protein band with molecular weight of 27 kDa was observed on SDS-PAGE gel. The recombinant protein was then purified through affinity chromatography, and the purity is more than 95%. The amino acid sequence of the recombinant protein was validated by NanoLC-MS/MS. The data from in vitro BIAcore analysis showed that the recombinant PSMB1 could bind to celastrol. The binding affinity between PSMB1 and 10 micromol/L celastrol was more than 27RU.
Subject(s)
Humans , Binding Sites , Escherichia coli , Genetics , Metabolism , Genetic Vectors , Genetics , Proteasome Endopeptidase Complex , Genetics , Proteasome Inhibitors , Recombinant Proteins , Genetics , Metabolism , Triterpenes , Metabolism , UbiquitinABSTRACT
<p><b>OBJECTIVE</b>To seek possible effect targets of bufalin in HeLa cells by studying the impact of bufalin on cell protein expression profile after treatment on human cervical carcinoma cell lines HeLa.</p><p><b>METHOD</b>Bufalin's ICs0was measured by MTr assay. The apoptosis of cells was observed by FCM (flow cytometry) and Hoechst 33342 staining assay. Differentiated expression protein spots were founded and identified using proteomic techniques, which could induce HeLa cell apoptosis.</p><p><b>RESULT</b>Bufalin showed remarkable cytotoxic effect on HeLa cells. IC50 (154 +/- 21.5) nmol X L(-1) indicated the possibility of inducing cell apoptosis. The protein expression profile showed 11 differentiated expression protein spots. Among the 11 proteins, nudix-type motif 5, vimentin, hnRNP C1/hnRNP C2 variant, HNRPK, HNRPK isoform a variant (two spots are the same protein), heat shock protein 27, macrophage-capping protein, SELENBP1 protein were down-regulated, while ribosomal protein, large, P0 and S-adenosylmethionine synthetase 2 were up-regulated by bufalin treatment. They may be effect targets of bufalin in HeLa cells. Western blotting showed consistent results in heat shock protein 27, vimentin and HNRPK between expression after treatment with bufalin and two-dimensional electrophoresis.</p><p><b>CONCLUSION</b>Bufa-Lin can induce apoptosis in human cervical carcinoma cells HeLa and the effect of bufalin may be related to the joint intervention with multiple protein targets.</p>
Subject(s)
Female , Humans , Antineoplastic Agents , Pharmacology , Apoptosis , Bufanolides , Pharmacology , Cell Line, Tumor , HeLa Cells , Neoplasm Proteins , Metabolism , Proteomics , Methods , Uterine Cervical Neoplasms , Drug Therapy , Metabolism , PathologyABSTRACT
Objective The oligomeric and polymeric proanthocyanidins in different fractions from Hippophae rhamnoides seeds were investigated. Methods The monomeric and oligomeric fractions were analyzed by HPLC/ESI-MASS detection. The polymeric proanthocyanidins were fractionated by Sephadex LH-20 column chromatography and their chemical constructures were studied by acid-catalysed degradation in the presence of benzyl mercaptan. Results Four monomers were identified as catechin, epicatechin, gallocatechin, and epigallocatechin. Eight dimers including three dimeric procyanidins and one dimeric prodelphinidin, and four mixed dimers were detected and their structures were partly elucidated by the detection of the pseudomolecular ion and main ion fragmentations. The results also showed that polymers to be highly heterogenous with catechin, epicatechin, gallocatechin, and epigallocatechin all being components of both the extension and the terminal units. The extension units were mainly gallocatechin, whereas the terminal units were mainly gallocatechin and epigallocatechin for all fractions. The proanthocyanidins with a mean degree of polymerization (mDP) of 9.1, 13.2 and 17.0 represented the three major polymeric fractions. Conclusion Proanthocyanidins of H. rhamnoides seeds, predominantly being of prodelphinidin-type, are remarkably different from that of grape seeds.