RESUMEN
Heparinase III is an enzyme that specifically cleaves certain sequences of heparan sulfate. Previous reports showed that this enzyme expressed in Escherichia coli was highly prone to aggregation in inclusion bodies and lacks detectable biological activity. In this paper, we fused a glutathione-S-transferase (GST) tag to the N-terminus of heparinase III gene and expressed the fusion protein in Escherichia coli to develop an expression system of soluble heparinase III. As a result, approximately 80% of the fusion protein was soluble. The protein was then purified to near homogeneity via one-step affinity chromatography. A 199.4-fold purification was achieved and the purified enzyme had a specific activity of 101.7 IU/mg protein. This represented 32.3% recovery of the total activity of recombinant GST-heparinase III. The maximum enzyme production was achieved when bacteria were induced with 0.5 mmol/L isopropyl-beta-D-thiogalactoside at 15 degrees C for 12 h. The enzyme showed maximum activity at 30 degrees C and pH 7.5. And the enzyme activity was stimulated by 1 mmol/L Ca2+ and 150 mmol/L NaCl.
Asunto(s)
Flavobacterium/enzimología , Glutatión Transferasa/biosíntesis , Liasa de Heparina/biosíntesis , Escherichia coli/genética , Escherichia coli/metabolismo , Flavobacterium/genética , Flavobacterium/crecimiento & desarrollo , Glutatión Transferasa/genética , Liasa de Heparina/genética , Liasa de Heparina/aislamiento & purificación , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificaciónRESUMEN
Manganese superoxide dismutase (MnSOD) is the only primary antioxidant enzyme in mitochondria that scavenges superoxide radicals. Overexpressing MnSOD in cancer cells by cDNA transfection suppresses tumor formation and reverses malignant growth. In this study, we examined the effect of recombinant human manganese superoxide dismutase (rhMnSOD) alone and in combination with adriamycin (ADR) against solid tumors of sarcoma 180 in Institute of Cancer Research (ICR) mice. Administration of rhMnSOD alone and in combination with ADR significantly inhibited tumor growth in a dose-dependent manner. The use of rhMnSOD in combination with ADR enhanced ADR's anti-tumor potency without increasing toxicity. Histopathological examination provided evidence of the anti-tumor effect. In addition, we found lymphocyte infiltration of the tumors, with an increase in both CD4- and CD8-positive cells in the treated tumors. The expression of CD4 and CD8 was up-regulated with increasing dose of rhMnSOD, and the combination treatment with ADR further enhanced this up-regulation. Collectively, these data indicate that rhMnSOD may exhibit an anti-tumor effect by stimulating the immune system and promoting the recruitment of lymphocytes into the tumor to kill tumor cells. Thus MnSOD may constitute a potential new therapeutic agent to be exploited as an adjuvant in cancer therapy.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica , Doxorrubicina/uso terapéutico , Proteínas Recombinantes/uso terapéutico , Sarcoma 180/tratamiento farmacológico , Superóxido Dismutasa/uso terapéutico , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Modelos Animales de Enfermedad , Humanos , Linfocitos Infiltrantes de Tumor , Ratones , Sarcoma 180/inmunología , Sarcoma 180/patologíaRESUMEN
OBJECTIVE: Investigating the antioxidant activities of water and ethanol extracts of natural Cordyceps sinensis and Cordyceps militaris and their fermentation preparations. METHOD: The samples were tested through 6 assays: inhibition ability of linoleic acid oxidation; scavenging activity of DPPH, hydrogen peroxide, hydroxyl radical and superoxide anion; and metal chelating activity. RESULT: Samples showed different antioxidant ability, and there was not an extract that exhibited high activity in all assays; however, water extract of natural C. militaris could be regarded as the most powerful antioxidant among 8 samples. It had high activity in inhibition of linoleic acid oxidation, chelating metal ions, and scavenging DPPH and hydroxyl radical. The research also indicated that the contents of phenolic compounds in water and ethanol extracts of natural and cultured Cordyceps sp. had huge difference. CONCLUSION: Natural Cordyceps sp. and its fermentation preparations could be used as potential natural antioxidants. The fermented process affected the antioxidant ability of cultured Cordyceps sp., and the antioxidant activity of both natural and cultured Cordyceps sp. did not significantly related with the quantity of phenolics.
Asunto(s)
Antioxidantes/farmacología , Cordyceps/química , Depuradores de Radicales Libres/farmacología , Materia Medica/farmacología , Antioxidantes/aislamiento & purificación , Quelantes/aislamiento & purificación , Quelantes/farmacología , Cordyceps/crecimiento & desarrollo , Cordyceps/metabolismo , Etanol , Fermentación , Flavonoides/análisis , Flavonoides/metabolismo , Depuradores de Radicales Libres/aislamiento & purificación , Ácido Linoleico/metabolismo , Materia Medica/aislamiento & purificación , Oxidación-Reducción/efectos de los fármacos , Fenoles/análisis , Fenoles/metabolismo , PolifenolesRESUMEN
Nineteen mutants of decaprenyl diphosphate synthase from Gluconobacter suboxydans were generated and their production of ubiquinones (UQs) was compared to that of the wild type protein. The accessible surface area and the polarity of amino acid around the catalytic pocket wield remarkable influences on the isoprenoid chain elongation of UQs.
Asunto(s)
Transferasas Alquil y Aril/química , Transferasas Alquil y Aril/metabolismo , Aminoácidos/metabolismo , Terpenos/metabolismo , Ubiquinona/biosíntesis , Transferasas Alquil y Aril/genética , Aminoácidos/química , Aminoácidos/genética , Secuencia de Bases , Sitios de Unión , Catálisis , Cromatografía Líquida de Alta Presión , Mutación/genética , Estructura Terciaria de Proteína , Especificidad por SustratoRESUMEN
Heparinase I has been purified from F. heparinum by a novel scheme with 10mM CaCl(2) added in crude extracts of cells. The enzyme was purified to apparent homogeneity through ammonium sulfate precipitation, Octyl-Sepharose chromatography, CM-52 chromatography, SP-650 chromatography, and Sephadex G-100 gel filtration chromatography. The specific activity of the purified enzyme was 90.33 U/mg protein with a purification fold of 185.1. The yield was 17.8%, which is higher than any previous scheme achieved. The molecular weight of the purified enzyme was 43 kDa with a pI of 8.5. It has an activity maximum at pH range of 6.4-7.0 and 41 degrees C. CaCl(2) is a good stabilizer of the purified enzyme in liquid form toward either storaging at 4 degrees C or freezing-thawing.
Asunto(s)
Cloruro de Calcio/farmacología , Cromatografía en Gel/métodos , Flavobacterium/enzimología , Liasa de Heparina/aislamiento & purificación , Estabilidad de Enzimas/efectos de los fármacos , Liasa de Heparina/efectos de los fármacos , Concentración de Iones de Hidrógeno , TemperaturaRESUMEN
B lymphocyte stimulator (BLyS), a member of the tumor necrosis factor superfamily of ligands, is a crucial survival factor for B cells. We successfully constructed seven mutants of the functional soluble fragment of human BLyS (named cBLyS, amino acid 134-285), including three deletion mutants and four site-directed mutants. All the mutant proteins were expressed in Escherichia coli and purified by Ni-NTA affinity chromatography. The biological activities of these mutants were assessed by the ligand-receptor binding assay, B cell proliferation assay and immune effect response in vivo. Our results indicated that four residues, H218, F220, T228 and L229, are indispensable for the biological activity of cBLyS, whereas two regions, amino acid 134-148 and amino acid 271-285, are related to the biological activity of BLyS. The protein of deletion of amino acid 134-148 leads to a complete defection in raising the antigen-specific IgM titer. The deletion of amino acid 271-285 reduces the effectiveness compared with the native cBLyS. This indicates that the region of amino acid 134-148 is indispensable for cBLyS to function normally.
Asunto(s)
Proteínas de la Membrana/genética , Factor de Necrosis Tumoral alfa/genética , Secuencia de Aminoácidos , Animales , Factor Activador de Células B , Antígeno de Maduración de Linfocitos B , Cromatografía de Afinidad , Femenino , Eliminación de Gen , Humanos , Inmunoglobulina G/biosíntesis , Inmunoglobulina M/biosíntesis , Proteínas de la Membrana/química , Proteínas de la Membrana/fisiología , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Mutación , Reacción en Cadena de la Polimerasa , Receptores del Factor de Necrosis Tumoral/metabolismo , Factor de Necrosis Tumoral alfa/química , Factor de Necrosis Tumoral alfa/fisiologíaRESUMEN
Effect of carbon, nitrogen, and metal ion sources on superoxide dismutase (SOD), catalase (CAT) activities, and lipid perioxide (LPO) levels in Cordyceps militaris mycelium were investigated at stationary growth phase by step supplementing with these nutrition factors in shake-flask cultures. Mycelium was cultivated in several growth media containing different carbon sources. The observed highest SOD and CAT activities were 44.3 U/mg protein in the presence of 20% potato broth plus 2% glucose medium and 93.7 U/mg protein in presence of 20% potato broth plus 1% glucose medium, respectively. By supplementing with either yeast extract or tryptone in 0.1-0.5% concentration range, the highest SOD and CAT activities were 21.1 U/mg protein in medium supplemented with 0.1% yeast extract and 20.7 U/mg protein in medium supplemented with 0.1% tryptone, respectively. Supplementing with Cu(2+), Zn(2+), and Mn(2+) caused a stimulation of SOD synthesis. The minimum LPO level was observed at media presented Zn(2+). The time course of SOD and CAT biosynthesis showed two maxima, which correspond to the maximum of biomass. High SOD levels and low LPO levels in the medium described above indicated that the appropriate metal ions could provide a suitable protection for cells against oxygen radical damage.
Asunto(s)
Catalasa/biosíntesis , Cordyceps/metabolismo , Peróxidos Lipídicos/metabolismo , Micelio/enzimología , Superóxido Dismutasa/biosíntesis , Biomasa , Carbono/metabolismo , Cordyceps/enzimología , Medios de Cultivo/química , Metales/metabolismo , Nitrógeno/metabolismo , Factores de TiempoRESUMEN
A decaprenyl diphosphate synthase gene (ddsA, GenBank accession No. DQ191802) was cloned from Rhodobacter capsulatus B10 by constructing and screening the genome library. An open reading frame of 1002 bp was revealed from sequence analysis. The deduced polypeptide consisted of 333 amino acids residues with an molecular mass of about 37 kDa. The DdsA protein contained the conserved amino acid sequence (DDXXD) of E-type polyprenyl diphosphate synthase and showed high similarity to others. In contrast, DdsA showed only 39% identity to a solanesyl diphosphate synthase cloned from R. capsulatus SB1003. DdsA was expressed successfully in Escherichia coli. Assaying the enzyme in vivo found it made E.coli synthesize UQ-10 in addition to the endogenous production UQ-8.
Asunto(s)
Transferasas Alquil y Aril/genética , Transferasas Alquil y Aril/metabolismo , Rhodobacter capsulatus/enzimología , Rhodobacter capsulatus/genética , Secuencia de Aminoácidos , Secuencia de Bases , Cromatografía Líquida de Alta Presión , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Escherichia coli/metabolismo , Datos de Secuencia Molecular , Proteínas Recombinantes/metabolismo , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Ubiquinona/metabolismoRESUMEN
The cDNA of Cu, Zn containing superoxide dismutase from the Cordyceps militaris SH (cm-SOD) was overexpressed in Escherichia coli BL 21 (DE3) using the pET-21a expression vector. The recombinant cell overexpressed the protein corresponding to 35+/-3% of total bacterial protein in cytosol. The purification was performed through three steps: DEAE-FF, CM-52, and G-100. After this purification procedure, a specific activity of 27272.7 U/mg of protein was reached, corresponding to 6.1-fold purification with a yield of 85.0%. The purity was homogeneous by SDS-PAGE analysis and 94.2+/-1.0% by CZE analysis. A subunit molecular mass of the recombinant enzyme was 15704 Da with a Cu and Zn element. In addition, the dimeric and polymeric structures were observed on MALDI-TOF-MS. Isoelectric point value of 7.0 was obtained for the recombinant enzyme that was sensitive to H2O2 and KCN. The recombinant enzyme remained 80+/-2% residual activity at pH 7.8, at 50 degrees C for 4h incubation. The properties: N-terminal amino acid sequence (the first 12 amino acid residues), pI, subunit molecular mass, thermo-stability of the purified recombinant SOD are similar to that of the native Cu, Zn-SOD from C. militaris (N-cm-SOD).
Asunto(s)
Cordyceps/enzimología , Superóxido Dismutasa/aislamiento & purificación , Secuencia de Aminoácidos , Electroforesis en Gel de Poliacrilamida , Estabilidad de Enzimas , Escherichia coli/enzimología , Peróxido de Hidrógeno/farmacología , Datos de Secuencia Molecular , Cianuro de Potasio/farmacología , Proteínas Recombinantes/aislamiento & purificación , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Superóxido Dismutasa/antagonistas & inhibidores , Superóxido Dismutasa/biosíntesisRESUMEN
This study was carried out to investigate whether shark hepatic stimulator substance (HSS) can prevent acute liver injury and affect mitochondrial function and antioxidant defenses in a rat model of thioacetamide (TAA)-induced liver injury. The acute liver injury was induced by two intraperitoneal injections of TAA (400 mg/kg) in a 24 h interval. In the TAA plus shark HSS group, rats were treated with shark HSS (80 mg/kg) 1 h prior to each TAA injection. In this group, serum liver enzyme activities were significantly lower than those in the TAA group. The mitochondrial respiratory control ratio was improved, and the mitochondrial respiratory enzyme activities were increased in the TAA plus shark HSS group. The mitochondrial antioxidant enzyme activities and glutathione level were higher in the TAA plus shark HSS group than in the TAA group. These results suggest that the protective effect of shark HSS against TAA-induced acute liver injury may be a result of the restoration of the mitochondrial respiratory function and antioxidant defenses and decreased oxygen stress.
Asunto(s)
Modelos Animales de Enfermedad , Fallo Hepático Agudo/metabolismo , Fallo Hepático Agudo/prevención & control , Regeneración Hepática/efectos de los fármacos , Mitocondrias Hepáticas/metabolismo , Péptidos/administración & dosificación , Especies Reactivas de Oxígeno/metabolismo , Animales , Antioxidantes/metabolismo , Respiración de la Célula/efectos de los fármacos , Péptidos y Proteínas de Señalización Intercelular , Fallo Hepático Agudo/inducido químicamente , Masculino , Mitocondrias Hepáticas/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Tiburones/metabolismo , Tioacetamida , Resultado del TratamientoRESUMEN
The EC-SOD cDNA was cloned by polymerase chain reaction (PCR) and inserted into the Escherichia coli expression plasmid pET-28a(+) and transformed into E. coli BL21(DE3). The corresponding protein that was overexpressed as a recombinant His6-tagged EC-SOD was present in the form of inactive inclusion bodies. This structure was first solubilized under denaturant conditions (8.0 M urea). Then, after a capture step using immobilized metal affinity chromatography (IMAC), a gradual refolding of the protein was performed on-column using a linear urea gradient from 8.0 M to 1.5 M in the presence of glutathione (GSH) and oxidized glutathione (GSSG). The mass ratio of GSH to GSSG was 4:1. The purified enzyme was active, showing that at least part of the protein was properly refolded. The protein was made concentrated by ultrafiltration, and then isolated using Sephacryl S-200 HR. There were two protein peaks in the A280 profile. Based on the results of electrophoresis, we concluded that the two fractions were formed by protein subunits of the same mass, and in the fraction where the molecular weight was higher, the dimer was formed through the disulfide bond between subunits. Activities were detected in the two fractions, but the activity of the dimer was much higher than that of the single monomer. The special activities of the two fractions were found to be 3475 U/mg protein and 510 U/mg protein, respectively.
Asunto(s)
Escherichia coli/enzimología , Escherichia coli/metabolismo , Superóxido Dismutasa/biosíntesis , Superóxido Dismutasa/química , Secuencia de Aminoácidos , Cromatografía de Afinidad/métodos , Histidina , Humanos , Datos de Secuencia Molecular , Peso Molecular , Proteínas Recombinantes/análisis , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Solubilidad , Superóxido Dismutasa/análisis , Superóxido Dismutasa/genéticaRESUMEN
Copper-zinc superoxide dismutase (Cu,Zn SOD) has been extracted, purified and characterized from Radix lethospermi seed (RLS), a kind of Chinese traditional medicine. Before extraction, the lipid was removed by super critical fluid extraction (SCF). Partial protein fractionation in the crude extract was affected by using 50-75% (NH(4))(2)SO(2). Subsequently, superoxide dismutase was fractionated by column chromatographies on DEAE-52, Sephadex G-200 and DEAE-52 again. Pure Cu,Zn SOD had a specific activity of 4843 U/mg protein and was purified 267.2-fold, with a yield of 23.55%. The purified enzyme has a molecular weight of about 30,500+/-100 and is composed of two non-covalently joined equal subunits. Purity was confirmed by Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), HPLC and mass spectroscopy. Amino acid content has been investigated. The enzyme was found to remain stable in the pH range 6.0-9.0 at 25 degrees C and up to 45 degrees C at pH 7.8 for a 30 min incubation period. RLS Cu,Zn SOD appeared to have significant thermal stability lower than other Cu,Zn SODs, as revealed by irreversible heat inactivation at 60 degrees C. The enzyme was not inhibited by DTT, NaN(3) and beta-mercaptoethanol, but was inhibited by cyanide and hydrogen peroxide. Finally, in the presence of 2 mM ethylendiamine tetra acetic acid (EDTA) and sodium dodecyl sulphate (SDS), the enzyme showed approximately 18 and 34% activity loss.
Asunto(s)
Plantas Medicinales/enzimología , Semillas/enzimología , Superóxido Dismutasa/aislamiento & purificación , Aminoácidos/análisis , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Ácido Edético/farmacología , Electroforesis en Gel de Poliacrilamida , Estabilidad de Enzimas , Peróxido de Hidrógeno/farmacología , Concentración de Iones de Hidrógeno , Cianuro de Potasio/farmacología , Espectrofotometría Ultravioleta , Superóxido Dismutasa/antagonistas & inhibidores , Superóxido Dismutasa/química , TemperaturaRESUMEN
A fusion thymosin alpha1-soluble B lymphocyte stimulator (TM alpha1-cBLyS) gene was generated to engineer a bifunctional lymphokine, which was then over-produced in Escherichia coli. The molecular weight of the expressed fusion protein was approximately 28 kDa. After being purified by Ni-NTA affinity column, the fusion protein had full activity of BLyS with a slightly higher immunological action than synthetic TMalpha1. Because TM alpha1 regulates the cellular immune response and cBLyS amplifies the humoral response, this bifunctional lymphokine could be useful in the treatment of various immunodeficiency syndromes and serve as an immunomodulator to enhance the host's response to vaccination.
Asunto(s)
Escherichia coli/metabolismo , Proteínas de la Membrana/metabolismo , Fragmentos de Péptidos/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Timosina/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Factor Activador de Células B , Linfocitos B/citología , Linfocitos B/efectos de los fármacos , Western Blotting , Proliferación Celular/efectos de los fármacos , Escherichia coli/genética , Femenino , Expresión Génica , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos BALB C , Muramidasa/inmunología , Fragmentos de Péptidos/genética , Unión Proteica , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/farmacología , Formación de Roseta , Ovinos , Linfocitos T/citología , Linfocitos T/inmunología , Timosina/genética , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
A gene fragment encoding three copies of proinsulin C-peptide was synthesized and expressed in E. coli and the recombinant proinsulin C-peptide was produced through site-specific cleavage of the resulting gene products. The fusion protein was expressed at high level, about 80 mg/L, as a soluble product in the cytoplasm. Ni-NTA affinity chromatography efficiently separated the expressed fusion protein from the supernatant, to obtain about 37.5 mg/L of the fusion protein with 70% purity. Enzymatic digestion by trypsin and carboxypeptidase B of the fusion protein efficiently released native C-peptide, the overall yield of recombinant C-peptide at a purity over 95% was 1.5 mg/L. The good agreement of amino acids composition, together with shown similarities of the recombinant C-peptide to C-peptide standard in the comparative RP-HPLC analysis and IMMULITE C-Peptide quantitative assay, suggested that the recombinant C-peptide obtained in this report was the native human C-peptide. The investigation of the chemical stability of recombinant human C-peptide in aqueous solutions by RP-HPLC was also reported. The degradation of the recombinant C-peptide showed a marked dependence on pH and temperature. The degradation reaction of C-peptide occurred immediately in pH 3 or pH 9 buffered solution. The degradation reaction of C-peptide followed first-order kinetics in pH 3 buffered solution at 37 degrees C or 70 degrees C, only 40.3% of C-peptide was remained after 10 h at 70 degrees C. The maximum stability was achieved at pH 7.4, more than 90% of C-peptide were detected at pH 7.4 and 37 degrees C after 10 h and at pH 7.4 and 70 degrees C after 5 h. 99% and 96% of C-peptide was remained at pH 7.4 and 37 degrees C after 10 h with and without 10 g/L BSA respectively.
Asunto(s)
Péptido C/metabolismo , Escherichia coli/genética , Secuencia de Aminoácidos , Péptido C/química , Péptido C/genética , Cromatografía de Afinidad/métodos , Cromatografía Líquida de Alta Presión/métodos , Electroforesis en Gel de Poliacrilamida , Expresión Génica , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Soluciones , Temperatura , Factores de TiempoRESUMEN
Recombinant bacteria exhibiting high enzymatic activities were obtained by cloning and coexpression of both caiB gene and caiE gene in the host of E. coli Bl21(DE3), which encode carnitine dehydratase and a protein related to the synthesis of cofactor for carnitine dehydratase, respectively. In order to coexpress these two genes, compatible and incompatible two plasmids system were used, the difference between them was also studied. After induction with IPTG, both caiB and caiE genes were coexpressed in both compatible two plasmids system and incompatible two plasmids system. In the former system, the expressed products accounted for 17% and 10% of the total proteins in the host; in the later system, the proportion was 39% and 20%, respectively. The activity of carnitine dehydratase in E. coli Bl21(DE3) with either coexpression system is about 2.3 times than that in E. coli Bl21(DE3) with only pET28-caiB. The plasmids stabilities in these two systems were the same, all needed the help of antibiotic selective pressure.
Asunto(s)
Aciltransferasas/biosíntesis , Proteínas Bacterianas/biosíntesis , Escherichia coli/metabolismo , Escherichia coli/enzimología , Escherichia coli/genética , Expresión Génica , Plásmidos/genéticaRESUMEN
Much is known about the physical properties of the Cu,Zn- and Mn-superoxide dismutases (SODs). However, the biochemical characteristics and pharmacological properties of extracellular (EC)-SOD have been severely limited due to difficulties in obtaining and purifying the enzyme. The EC-SOD cDNA was inserted into the Escherichia coli expression plasmid pET-28a(+) which contains the T7 promoter and transformed into the E. coli BL21(DE3). After induction with 1 mmol/L isopropyl beta-D-thiogalactoside, the recombinant human EC-SOD was highly expressed as inclusion bodies. SDS-PAGE analysis revealed that recombinant EC-SOD accumulated up to 26% of the total soluble protein of E. coli cells. The expression product was purified by a Ni(2+)-IDA-Sepharose 6B column. After the denaturing and refolding processes, the recombinant human EC-SOD retains the specific enzymatic activity of 920 U/mg of the purified product. The gene encoding human EC-SOD mature peptide was also inserted into the donor plasmid pFastBacHTb. After transposition, transfection, and amplification were performed, the recombinant baculoviruses infected the Tn-5B1-4 cells and EC-SOD was highly expressed in Tn-5B1-4 cells. SDS-PAGE and Western blot analysis revealed that the subunit molecular weight of the expression product is 28 kDa. Furthermore, recombinant human EC-SOD retains the enzymatic specific activity of 200 U/mg of the Tn-5B1-4 cell lysates.
Asunto(s)
Clonación Molecular/métodos , Superóxido Dismutasa/genética , Animales , Baculoviridae , Células Cultivadas , Cromatografía de Afinidad , Cobre/química , Escherichia coli , Expresión Génica , Humanos , Cuerpos de Inclusión , Mariposas Nocturnas , Superóxido Dismutasa/química , Zinc/químicaRESUMEN
Oxygen-derived free radicals are thought to be involved in the pathogenesis of a wide range of neurological disorders. Targeted delivery of CuZn-SOD to neurons in central nervous system may have therapeutic value in such diseases. The gene encoding human CuZn-SOD was fused to tetanus toxin fragment C geneto construct a fusion gene, then it was cloned into prokaryotic expression vector pET-22b( ) and baculovirus vector pFastBacHTb, and was expressed in E.coli and Tn-5B1-4 cells, respectively. The recombinant fusion protein has a subunit molecular mass of 68 kD and is recognized by both anti-CuZn-SOD and anti-tetanustoxin antibody. By pyrogallol autooxidation assay, it is shown that the CuZn-SOD moiety retains substantial enzymatic activity, where the TTC moiety might deliver the fusion protein to neurons in central nervous system. So, CuZn-SOD/TTC may be a useful agent for the targeted delivery of CuZn-SOD to neurons.
RESUMEN
The fragment C of tetanus toxin was amplified from Clostridium tetani DNA by PCR. This fragment was cloned into expression vector pET-28a(+),under the control of the T7 promoter. Expression of this plasmid in E.coli resulted in the production of a protein consisting of 6xHis of the vector fused to the N-terminal 451 amino acids of tetanus toxin. After induction with 1 mmol/L IPTG, TTC was expressed in E.coli BL21(DE3). The protein product accounted for 8.2% of the bacteria total protein in soluble form, SDS-PAGE and Western blot analysis of TTC recombinant protein confirmed this result. The expression products were also purified by Ni(2+)-IDA-Sephrose 6B column. Immunization of mice with rTTC resulted in the production of antibodies that were able to protect mice against a challenge with tetanus toxin furthermore, rTTC in vivo appeared to be able to undergo retrograde axonal transport.
RESUMEN
The cDNA encoding human manganese-superoxide dismutase was amplified from the human liver cell (L02) total RNA by RT-PCR and sequenced. The human Mn-SOD cDNA was ligated into expression vector pET-24a(+) under T7 promoter. After 5 h induction with 1 mmol/L IPTG 800 mol/L Mn(2+) human Mn-SOD was highly expressed in E.coli BL21(DE3). The protein product was up to 50% of the bacteria total protein in soluble form and had specific SOD activity.
RESUMEN
The cDNA encoding the human copper, zinc-superoxide dismutase(Cu, Zn-SOD) was amplified from the human liver by RT-PCR and sequenced. The cloned human Cu, Zn-SOD cDNA was ligated into expression vector pET-22b(+) under T7 promotor. After 3 h induction with 1 mmol/L IPTG, human Cu, Zn-SOD was highly expressed in E.coli BL21(DE3). The expression product was up to 30% of the total protein of the bacteria in soluble form, which had specific SOD activity.