Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P00750 (PLA)
 16,800 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Dengue is one of the most important mosquito-borne viral disease causing dengue fever and/or dengue shock syndrome/haemorrhagic fever. In some reports, the non-structural protein 1 (NS1) has been identified as a promising antigen for the development of vaccines against dengue virus (DENV). Apparently, it can elicit a protective antibody response with complement-fixing activities. In order to investigate the potential of a DNA vaccine based on the NS1 protein against DENV, we used the plasmid pcTPANS1, which contains the secretory signal sequence derived from human tissue plasminogen activator (t-PA) fused to the full length of the DENV-2 NS1 gene. All Balb/c mice intramuscularly inoculated with the pcTPANS1 presented high levels of NS1-specifc antibodies. Vaccinated animals were challenged with intracerebral DENV-2 virus inoculations and a 100% survival was observed. In general, results demonstrate that the pcTPANS1 plasmid is able to induce protection in mice, and then may be used as a vaccination approach against DENV in further assays.
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PMID:DNA vaccine against the non-structural 1 protein (NS1) of dengue 2 virus. 1614 Apr 30

Recombinant human tissue-type plasminogen activator derivative (r-PA), fused with thioredoxin (Trx), was expressed in Escherichia coli. The resultant fusion protein, Trx-r-PA, was almost completely in the form of inclusion bodies and without activity. Different refolding strategies were investigated including different post-treatment of solubilized Trx-r-PA inclusion bodies, on-column refolding by size-exclusion chromatography (SEC) using three gel types (Sephacryl S-200, S-300 and S-400), refolding by Sephacryl S-200 with a urea gradient and two-stage temperature control in refolding. An optimized on-column refolding process for Trx-r-PA inclusion bodies was established. The collected Trx-r-PA inclusion bodies were dissolved in 6 M: guanidine hydrochloride (Gdm.HCl), and the denatured protein was separated from dithiothreitol (DTT) and Gdm.HCl with a G25 column and simultaneously dissolved in 8 M: urea containing oxidized glutathione (GSSG). Finally a refolding of Trx-r-PA protein on Sephacryl S-200 column with a decreasing urea gradient combined with two-stage temperature control was employed, and the activity recovery of refolded protein was increased from 3.6 to 13.8% in comparison with the usual dilution refolding.
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PMID:A comparative investigation on different refolding strategies of recombinant human tissue-type plasminogen activator derivative. 1661 26

In this study, fused deposition manufacturing (FDM) was utilized to fabricate the precision scaffolds for cartilage and bone regeneration. Cell seeding into such scaffolds was evaluated. For poly(D,l-lactide) (PLA) scaffolds used for cartilage regeneration, the structure with larger inner space, four direction stacking (4D) and small interval of fibers were better. Chondrocyte proliferated well with matrix accumulation in precision scaffolds coated with type II collagen at 4 weeks of in vitro culture. The seeding efficiency of osteoblasts in most polycaprolactone (PCL) scaffolds used for bone regeneration could arrive 50% of original cell seeding density, and the amount of cells in scaffolds increased to double fold after 2 weeks of in vitro culture. The histological cross-section also revealed proliferation and mineralization of osteoblasts among the PCL fibers. The results indicated that the highly porous and interconnected structure of precision scaffolds could benefit cell ingrowth.
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PMID:Evaluation of the growth of chondrocytes and osteoblasts seeded into precision scaffolds fabricated by fused deposition manufacturing. 1686 56

The cDNA encoding a protease of Perinereis aibuhitensis Grube (PPA) was cloned. The deduced amino acid sequence analysis showed that the protein had 49% identity to the C-terminal amino acid 169-246 of serine protease of Heterodera glycines. Northern blotting analysis indicated that the cDNA could hybridize with mRNA of approximately 260 bases isolated from the marine earthworm. The cDNA was amplified by polymerase chain reaction and cloned into pMAL-p2 to construct expression vector pMAL-PPA. pMAL-PPA was introduced into Escherichia coli BL21(DE3) and overexpression of PPA fused with maltose binding protein was achieved by isopropyl-beta-D-thiogalactopyranoside induction. The fusion protein was purified by affinity chromatography on an amylose resin column and ion-exchange chromatography on a diethylaminoethyl-Sepharose 4B column. Rabbits were immunized with the purified protein and antiserum was prepared. The antibody could react with a protein of approximately 9 kDa extracted from the marine earthworm as shown by Western blotting analysis. The activity analysis of the recombinant PPA suggested that it was probably a plasminogen activator.
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PMID:Isolation of a cDNA encoding a protease from Perinereis aibuhitensis Grube. 1689 76

We analyzed four DNA vaccines based on DENV-2 NS1: pcENS1, encoding the C-terminal from E protein plus the NS1 region; pcENS1ANC, similar to pcENS1 plus the N-terminal sequence from NS2a (ANC); pcTPANS1, coding the t-PA signal sequence fused to NS1; and pcTPANS1ANC, similar to pcTPANS1 plus the ANC sequence. The NS1 was detected in lysates and culture supernatants from pcTPANS1-, pcENS1- and pcENS1ANC-transfected cells and not in cells with pcTPANS1ANC. Only the pcENS1ANC leads the expression of NS1 in plasma membrane, confirming the importance of ANC sequence for targeting NS1 to cell surface. High levels of antibodies recognizing conformational epitopes of NS1 were induced in mice immunized with pcTPANS1 and pcENS1, while only few pcENS1ANC-inoculated animals presented detectable anti-NS1 IgG. Protection against DENV-2 was verified in pcTPANS1- and pcENS1-immunized mice, although the plasmid pcTPANS1 induced slight higher protective immunity. These plasmids seem to activate distinct patterns of the immune system.
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PMID:DNA vaccines against dengue virus based on the ns1 gene: the influence of different signal sequences on the protein expression and its correlation to the immune response elicited in mice. 1702 Jul 77

Capillary electrophoretic (CE) methods were used for the quantitative determination of model drugs [salbutamol sulphate (SS), sodium cromoglycate (SCG) and beclomethasone dipropionate (BDP)] in poly(D,L-lactic acid) (PLA) nanoparticles, which were prepared by the nanoprecipitation method. Zeta potential and size distribution of the nanoparticles were determined by electrophoretic mobility determinations and photon correlation spectroscopy, respectively. Interactions between the drugs, the PLA nanoparticles and the fused-silica capillary were investigated by electrokinetic capillary chromatography (EKC). A quantitative CE method was developed for salbutamol sulphate and sodium cromoglycate, and the linearity and repeatability of migration times, peak areas and peak heights were determined. Microemulsion electrokinetic chromatography was used for the quantitative determination of beclomethasone dipropionate. According to this study, the applied electromigration techniques were suitable for the interaction, drug entrapment and dissolution studies of pharmaceutical nanoparticles. The results suggest that even quantitation of the drug located inside the nanoparticles was possible. Encapsulation of the more hydrophilic model drugs (SS, SCG) in the PLA nanoparticles was less efficient than in the case of BDP.
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PMID:Quantitative determination of drug encapsulation in poly(lactic acid) nanoparticles by capillary electrophoresis. 1805 45

A novel recombinant expression system in Escherichia coli was developed using conger eel galectin, namely, congerin II, as an affinity tag. This system was applied for the functional expression of myotoxic lysine-49-phospholipase A(2) ([Lys(49)]PLA(2)), termed BPII and obtained from Protobothrops flavoviridis (Pf) venom. Recombinant Pf BPII fused with a congerin tag has been successfully expressed as a soluble fraction and showed better quantitative yield when folded correctly. The solubility of the recombinant congerin II-tagged BPII increased up to >90% in E. coli strain JM109 when coexpressed with the molecular chaperones GroEL, GroES, and trigger factor (Tf). The tag protein was cleaved by digestion with restriction protease, such as alpha-thrombin or Microbacterium liquefaciens protease (MLP), to obtain completely active recombinant BPII. Thus, the congerin-tagged fusion systems containing the cleavage recognition site for alpha-thrombin or MLP were demonstrated to be highly efficient and useful for producing proteins of desired solubility and activity.
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PMID:A novel recombinant system for functional expression of myonecrotic snake phospholipase A(2) in Escherichia coli using a new fusion affinity tag. 1820 18

Conjugation or deconjugation of ubiquitin (Ub) or ubiquitin-like proteins (UBLs) to or from cellular proteins is a multifaceted and universal means of regulating cellular physiology, controlling the lifetime, localization, and activity of many critical proteins. Deconjugation of Ub or UBL from proteins is performed by a class of proteases called isopeptidases. Herein is described a readily quantifiable novel isopeptidase assay platform consisting of Ub or UBL fused to the reporter enzyme phospholipase A(2) (PLA(2)). Isopeptidase activity releases PLA(2), which cleaves its substrate, generating a signal that is linear with deubiquitylase (DUB) concentration and is able to discriminate DUB, deSUMOylase, deNEDDylase, and deISGylase activities. The power and sensitivity of the UBL-PLA(2) assay are demonstrated by its ability to differentiate the contrasting deISGylase and DUB activities of two coronavirus proteases: severe acute respiratory syndrome papain-like protease (SARS-CoV PLpro) and NL63 CoV papain-like protease 2 (PLP2). Furthermore, direct comparisons with the current Ub-7-amino-4-methylcoumarin (Ub-AMC) assay demonstrated that the Ub-PLA(2) assay is an effective tool for characterizing modulators of isopeptidase activity. This observation was expanded by profiling the inhibitory activity of the nonselective isopeptidase inhibitor NSC 632839 against DUBs and deSUMOylases. Taken together, these studies illustrate the utility of the reporter-based approach to measuring isopeptidase activity.
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PMID:Characterization of ubiquitin and ubiquitin-like-protein isopeptidase activities. 1842 14

A long-lasting recombinant human albumin-linker-erythropoietin (EPO) is a human albumin gene fused to the N-terminal of EPO with a (GGSGG)(n)-repeated linker inserted between albumin and EPO. Albumin-EPO fusion genes were co-transfected with the dhfr gene. Albumin-EPO fusion protein has three kinds of sub-types (IALE, AD2LE, AD1LE). Albumin-EPO fusion protein was quantified with human EPO ELISA. The in vitro efficacy of albumin-EPO fusion protein was estimated using F-36E cell, and in vivo efficacy of albumin-EPO fusion protein was estimated using normocythemic mice (B6D2F1). We also determined the in vivo half-life in a Sprague-Dawley rat. A PLA program analysis result demonstrated that the albumin-EPO fusion protein IALE is about 7.8-fold more potent than rHuEPO in increasing the hematocrit of normal mice.
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PMID:Production and characterization of long-acting recombinant human albumin-EPO fusion protein expressed in CHO cell. 1959 70

To establish a refolding process for the protein fused with 12-peptide of hirudin and reteplase (HV12p-rPA), we developed an anion-exchange chromatography assisted method to form correct disulfide bonds. After evaluating various parameters by orthogonal experiments with Q Sepharose XL as refolding medium, we found that urea gradient, sample loading size and L-Arg concentration were three major factors to affect the refolding outcomes, and urea gradient was critical to the recovery yield. Meanwhile, enzymatic activity of the refolded protein was decreased by the increase of sample loading size, and the optimal concentration of L-Arg in the eluting buffer was 1 mol/L. Thus, a dual-gradient of urea and pH on the anion-exchange chromatography resulted in remarkable increase of specific fibrinolytic and anti-coagulative activities of the refolded protein. Compared with the dilution method for refolding HV12p-rPA, the present approach was more effective and advantageous.
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PMID:[Chromatography-assisted refolding of a fusion protein containing multiple disulfide bonds]. 2109 Jan 23


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