Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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Immunotherapy of rheumatoid arthritis (RA) using oral-dosed native chicken or bovine type II collagen (nCII) to induce specific immune tolerance is an attractive strategy. However, the majority of clinical trials of oral tolerance in human diseases including RA in recent years have been disappointing. Here, we describe a novel recombinant peptide rcCTE1-2 which contains only two tolerogenic epitopes (CTE1 and CTE2) of chicken type II collagen (cCII). These are the critical T-cell determinants for suppression of RA that were first developed and used to compare its suppressive effects with ncCII on the collagen-induced arthritis (CIA) model. The rcCTE1-2 was produced using the prokaryotic pET expression system and purified by Ni-NTA His affinity chromatography. Strikingly, our results showed clearly that rcCTE1-2 was as efficacious as ncCII at the dose of 50 microg/kg/d. This dose significantly reduced footpad swelling, arthritic incidence and scores, and deferred the onset of disease. Furthermore, rcCTE1-2 of 50 microg/kg/d could lower the level of anti-nCII antibody in the serum of CIA animals, decrease Th1-cytokine INF-gamma level, and increase Th3-cytokine TGF-beta(1) produced level by spleen cells from CIA mice after in vivo stimulation with ncCII. Importantly, rcCTE1-2 was even more potent than native cCII, which was used in the clinic for RA. Equally importantly, the findings that the major T-cell determinants of cCII that are also recognized by H-2(b) MHC-restricted T cells have not previously been reported. Taken together, these results suggest that we have successfully developed a novel recombinant peptide rcCTE1-2 that can induce a potent tolerogenic response in CIA.
Mol Immunol 2009 Feb
PMID:A novel recombinant peptide containing only two T-cell tolerance epitopes of chicken type II collagen that suppresses collagen-induced arthritis. 1904 Nov 37

Small ubiquitin-related modifier (SUMO) fusion system has been shown to be efficient for enhancing expression and preventing degradation of the target protein. We showed herein that SUMO fusion to human keratinocyte growth factor 2 (hKGF-2) gene was feasible and it significantly enhanced protein expression and its efficiency. The fusion DNA fragment composed of SUMO gene, which was fused to hexahistidine tag, and hKGF-2 gene was amplified by PCR and inserted into the expression vector pET28a to construct the recombinant plasmid, pET28a-SUMO-hKGF-2. The plasmid was then transformed into Escherichia coli Rosetta(TM)2(DE3), and the recombinant fusion protein SUMO-hKGF-2 was expressed at 30 degrees C for 6 h, with the induction of IPTG at the final concentration of 0.4 mM. The expression level of the fusion protein was up to 30% of the total cellular protein. The fusion protein was purified by Ni-NTA affinity chromatography. After desalting by Sephadex G-25 size exclusion chromatography, the hexahistidine-SUMO-hKGF-2 was digested by SUMO proteases. The recombinant hKGF-2 was purified again with Ni-NTA column and the purity was about 95% with a total yield of 13.9 mg/l culture. The result of mitogenicity assay suggests that the recombinant hKGF-2 can significantly promote the proliferation of normal rat kidney epithelial (NRK-52E) cells.
Mol Biotechnol 2009 May
PMID:Expression and purification of human keratinocyte growth factor 2 by fusion with SUMO. 1910 60

Ni-NTA affinity chromatography under denaturing conditions has proven to be a powerful method for the isolation of SUMO conjugates from total cell extracts, as it minimizes deconjugation and excludes noncovalent interactions. This chapter describes the use of both His-tagged SUMO and a His-tagged target protein for the characterization of the sumoylation process in the budding yeast Saccharomyces cerevisiae. Two well-studied model substrates, the septin Cdc3 and the replication clamp protein PCNA, are used as examples, but the protocol can easily be adapted to other targets and organisms.
Methods Mol Biol 2009
PMID:In vivo detection and characterization of sumoylation targets in Saccharomyces cerevisiae. 1910 12

Curcumin (diferuloylmethane), a biologically active ingredient derived from rhizome of the plant Curcuma longa, has potent anticancer properties as demonstrated in a plethora of human cancer cell lines/animal carcinogenesis model and also acts as a biological response modifier in various disorders. We have reported previously that dietary supplementation of curcumin suppresses renal ornithine decarboxylase (Okazaki et al. Biochim Biophys Acta 1740:357-366, 2005) and enhances activities of antioxidant and phase II metabolizing enzymes in mice (Iqbal et al. Pharmacol Toxicol 92:33-38, 2003) and also inhibits Fe-NTA-induced oxidative injury of lipids and DNA in vitro (Iqbal et al. Teratog Carcinog Mutagen 1:151-160, 2003). This study was designed to examine whether curcumin possess the potential to suppress the oxidative damage caused by kidney-specific carcinogen, Fe-NTA, in animals. In accord with previous report, at 1 h after Fe-NTA treatment (9.0 mg Fe/kg body weight intraperitoneally), a substantial increased formation of 4-hydroxy-2-nonenal (HNE)-modified protein adducts in renal proximal tubules of animals was observed. Likewise, the levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG) and protein reactive carbonyl, an indicator of protein oxidation, were also increased at 1 h after Fe-NTA treatment in the kidneys of animals. The prophylactic feeding of animals with 1.0% curcumin in diet for 4 weeks completely abolished the formation of (i) HNE-modified protein adducts, (ii) 8-OHdG, and (iii) protein reactive carbonyl in the kidneys of Fe-NTA-treated animals. Taken together, our results suggest that curcumin may afford substantial protection against oxidative damage caused by Fe-NTA, and these protective effects may be mediated via its antioxidant properties. These properties of curcumin strongly suggest that it could be used as a cancer chemopreventive agent.
Mol Cell Biochem 2009 Apr
PMID:Curcumin attenuates oxidative damage in animals treated with a renal carcinogen, ferric nitrilotriacetate (Fe-NTA): implications for cancer prevention. 1916 75

UBE1 plays an important role in the first step of ubiquitin-proteasome pathway to activate ubiquitin. Both the structure and biochemical property research of human UBE1 protein, and the activity analysis of those enzymes which are related with ubiquitination pathway, are based on high purity of UBE1 protein. To obtain human UBE1 protein, the full length of human UBE1 was expressed in E. coli and purified by Ni-NTA superflow sepharose and strep-tactin sepharose which based on UB-UBE1 high-energy thioester bonded intermediate complex. It was demonstrated that purified UBE1 could activate and conjugate UB to ubiquitin-conjugating enzyme E2s. The purified large amount of UBE1 could be used for in vitro studies of ubiquitin pathway and structural studies.
Mol Biol Rep 2010 Mar
PMID:Expression, purification and characterization of human ubiquitin-activating enzyme, UBE1. 1934 38

A technique is described for the identification of nucleic acid sequences bound with high affinity by proteins or by other molecules suitable for a partitioning assay. Here, a histidine-tagged protein is allowed to interact with a pool of nucleic acids and the protein-nucleic acid complexes formed are retained on a Ni-NTA matrix. Nucleic acids with a low level of recognition by the protein are washed away. The pool of recovered nucleic acids is amplified by the polymerase chain reaction and is submitted to further rounds of selection. Each round of selection increases the proportion of sequences that are avidly bound by the protein of interest. The cloning and sequencing of these sequences finally completes their identification.
Methods Mol Biol 2009
PMID:Identification of nucleic acid high-affinity binding sequences of proteins by SELEX. 1937 65

The crystal structures of almost all the enzymes of arginine metabolism have been determined, but arginine decarboxylase's structure is not resolved yet. In order to characterize and crystallize arginine decarboxylase, we overexpressed biosynthetic arginine decarboxylase (ADC; EC 4.1.1.19, encoded by the speA gene) from Escherichia coli in the T7 expression system as a cleavable poly-His-tagged fusion construct. The expressed recombinant His(10)-ADC (77.3 kDa) was first purified by Ni-NTA affinity chromatography, then proteolytically digested with Tobacco Etch Virus (TEV) protease to remove the poly-His fusion tag, and finally purified by anion exchange chromatography. The His(10) tag removed recombinant ADC (74.1 kDa)'s typical yield was 90 mg from 1 l of culture medium with purity above 98%. The recombinant ADC was assayed for decarboxylase activity, showing decarboxylase activity of 2.8 U/mg, similar to the purified native E. coli ADC. The decarboxylase activity assay also showed that the purified recombinant ADC tolerated broad ranges of pH (pH 6-9) and temperature (20-80 degrees C). Our research may facilitate further studies of ADC structure and function, including the determination of its crystal structure by X-ray diffraction.
Mol Biol Rep 2010 Apr
PMID:Expression and purification of recombinant arginine decarboxylase (speA) from Escherichia coli. 1960 87

Although glucose-6-phosphate isomerase (GPI) plays an important role in glycolysis of both the prokaryotes and eukaryotes, studies on the GPI have not been involved in the halotolerant, unicellular green alga Dunaliella salina (D. salina). In this study, a 2,338 bp of full-length cDNA cloned using rapid amplification of cDNA end (RACE) technique contained an open reading frame (ORF) of 1,980 bp encoding 660 amino acids, which has a predicted molecular weight of 73.3 kD and pI of 6.22 and shares high homology with other organisms. The cloned full-length cDNA was heterologously expressed in Escherichia coli and the recombinant GPI proteins purified using Ni-NTA His Bind column were consistent with the anticipated size of ~75 kD. Predicted 2D and 3D structures of GPI proteins possessed potential active motifs including "GEPGTNGQHSFYQLIHQG" and "VQGFIWGINSFDQWGVELGK", and critical active site residues, such as Ser 241, Ser 296, Thr 298, Thr 301, Arg 358, Glu 444, His 475 and Lys 600. Real time quantitative RT-PCR demonstrated that the expression level of the GPI gene from D. salina (DsGPI) was induced by 3.5 M NaCl with 14-fold higher than that by 1.5 M NaCl (P < 0.01), but inhibited by the light with 4-fold lower than that in the dark (P < 0.05). It is concluded that the cloned GPI gene is indeed from D. salina and may respond to salt and light.
Mol Biol Rep 2010 Feb
PMID:Characterization of the glucose-6-phosphate isomerase (GPI) gene from the halotolerant alga Dunaliella salina. 1968 75

One of the concerns about influenza A vaccine based on M2e protein is their limited potency; hence, optimal approaches to enhance immunogenicity of M2e protein immunization remain to be established. It seems by linking this M2e-peptide to an appropriate carrier such as mycobacterium tuberculosis C-terminal 28-kDa domain of HSP70 (HSP70(359-610)), we can render it very immunogenic. According to previous reports, this study was designed to produce a novel influenza A virus recombinant fusion protein consisted of M2e, a potent immunogenic protein from influenza A virus, fused to C-terminal domain of mycobacterium tuberculosis HSP70, HSP70(359-610), as a carrier and adjuvant. We fused the genes of M2e and HSP70 ( 359-610 ) then inserted in pQE-60, prokaryotic expression vector. This recombinant fusion protein with a 6xHis-tag was successfully over expressed in Escherichia coli M-15. The recombinant fusion protein was purified by Ni-NTA affinity chromatography under denaturing conditions, followed by urea gradient dialysis. The purified fusion protein was analyzed on SDS-PAGE. Western blot assay was used to examine the immunoreaction of the expressed protein using commercial penta-His HRP conjugate antibody. The antigenicity and biological activity of the recombinant protein was also qualitatively detected on the infected MDCK cells surface by immunofluorescence and cell-ELISA assay using rabbit's immunized antiserum. This observation suggest that the expressed fusion protein is useful as a universal recombinant vaccine for overcoming highly mutational influenza virus, but more immunological study in animal lab remains to be evaluated.
Mol Biol Rep 2010 Jul
PMID:Heterologous expression, purification and characterization of the influenza A virus M2e gene fused to Mycobacterium tuberculosis HSP70(359-610) in prokaryotic system as a fusion protein. 1981 2

Human 17beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD1) catalyzes the reaction of estrone with NADPH to form estradiol and NADP(+), thereby regulating the biological activity of sex steroid hormones in a variety of tissues. Here, we present an efficient method for expressing and purifying human 17beta-HSD1 from Escherichia coli. The expression vector pET28a/17beta-HSD1 was constructed and transformed into Escherichia coli BL21(DE3) cells. We found that the active enzyme can be obtained by inducing 17beta-HSD1 expression at 0.25 mM IPTG, 13 degrees C for overnight. The protein is purified by single step Ni-NTA affinity chromatography and yields 2.8 mg/L of culture. The kinetic study shows V/E ( t ) of (1.21 +/- 0.05) x 10(-2)/s and K (estradiol) of 0.8 microM in the oxidation of estradiol with NADP(+) as cofactor at pH 9.3. The new bacterial expression system for recombinant 17beta-HSD1 is useful for the easy purification of large amounts and will facilitate the functional study of this enzyme.
Mol Biotechnol 2010 Feb
PMID:Expression, purification, and characterization of a human recombinant 17beta-hydroxysteroid dehydrogenase type 1 in Escherichia coli. 1991 84


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