Purification and properties of recombinant GST-heparinase III and optimization of cultivation conditions.

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.

Enhanced anti-tumor effects achieved in a murine tumor model using combination therapy of recombinant human manganese superoxide dismutase and adriamycin.

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.

[Antioxidant activity of natural and cultured Cordyceps sp].

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.

Cloning and characterization of the ddsA gene encoding decaprenyl diphosphate synthase from Rhodobacter capsulatus B10.

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.

Residues around the catalytic pocket of DdsA are important for isoprenoids chain elongation.

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.

Effect of CaCl2 as activity stabilizer on purification of heparinase I from Flavobacterium heparinum.

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.

Two observed regions in B lymphocyte stimulator important for its biological activity.

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.

Effect of nutrition factors on the synthesis of superoxide dismutase, catalase, and membrane lipid peroxide levels in Cordyceps militaris mycelium.

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.

Purification and partial characterization of recombinant Cu, Zn containing superoxide dismutase of Cordyceps militaris in E.coli.

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.

Effects of shark hepatic stimulator substance on the function and antioxidant capacity of liver mitochondria in an animal model of acute liver injury.

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.

On-column refolding of an insoluble His6-tagged recombinant EC-SOD overexpressed in Escherichia coli.

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.

Purification and some properties of Cu,Zn superoxide dismutase from Radix lethospermi seed, kind of Chinese traditional medicine.

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).

Construction and expression of a new fusion protein, thymosin alpha1-cBLyS, E. coli.

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.

Cloning and expression of a new rat procarboxypeptidase B gene in Escherichia coli and purification of recombination carboxypeptidase B.

A new coding sequence of the procarboxypeptidase B gene was obtained from SD rat fresh pancreas by RT-PCR and highly expressed in Escherichia coli in inclusion bodies. The folded procarboxypeptidase B was subjected to trypsin enzymatic cleavage to produce active carboxypeptidase B, subsequently, carboxypeptidase B was effectively purified with anion exchange chromatography DEAE-FF and hydrophobic interaction chromatography Octyl FF, as a result, 40 mg carboxypeptidase B per litre cell culture with specific activity 7.42 u/mg was achieved.

Expression of C-peptide multiple gene copies in Escherichia coli and stabilities of C-peptide in aqueous solution.

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.

Cloning, Sequencing and Expression of Human Copper, Zinc-superoxide Dismutase cDNA.

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.

Cloning and Expression of Human Manganese-superoxide Dismutase cDNA.

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.

Cloning and Expression of Tetanus Toxin Fragment C in E.coli.

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.

Cloning and Expression of Targeting CuZn-SOD to Central Nervous System.

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.

[Coexpression of caiB and caiE with two plasmids in E. coli].

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.

Structure Prediction and Analyzing of the Molecular Chaperone TCP1.

An improved Chou and Fasman method and the Robson method from Prosis software were applied to four TCP1s and five homologous proteins. The predictive results showed that there may exist two structural domains in TCPI with a "hinge region" between them, suggesting that the two structural domains may be alpha/beta barrels. The putative ATP-binding domain, whose motif centres around the absolute conserved GDGTT sequence at position 87-91 in TCP1Hu, was found to be a coil region in the predictive results.