cloning expression and purification of recombinant aldo keto reductases

... the classification of LASV as a National Institutes of Allergy and Infectious Diseases (NIAID) Category A pathogen and biosafety level-4 (BSL4) agent Expression and purification of E coli-generated ... 2(DE3) cells transformed with construct pMAL-c2x:NP Expression and purification of LASV NP from E coli Rosetta Expression and purification of LASV NP from E coli Rosetta 2(DE3) cells transformed ... transformed with construct from E coli Rosetta gami cellsand purification of LASV GP2pMAL-c2x:GP2 Expression Expression and purification of LASV GP2 from E coli Rosetta gami cells transformed with...

Ngày tải lên: 20/06/2014, 01:20

... describe the cloning and expression of the M tuberculosis tps gene in E coli, and the production of active TPS in these cells The properties of the recombinant enzyme have been determined, and are ... Occurrence of trehalose and molecular cloning and characterization of trehalose-6-P synthase homologues J Exp Bot 52, 1817–1826 Zaragoza, O., Blazquez, M.A & Gancedo, C (1998) Disruption of the Candida ... M) of NH4HCO3 An aliquot of each fraction was removed and assayed for its radioactive content The radioactive peak was pooled and concentrated to dryness a number of times in the presence of...

Ngày tải lên: 17/03/2014, 10:20

... as the amount of enzyme that catalyzes the oxidation of μmol of substrate per minute Results Cloning, expression and purification of 2, 5-diketocamphane 1,2-monooxygenase The 2,5-diketocamphane ... was introduced using the sites of restriction endonucleases NdeI and XhoI for cloning with the BCA-kit and a standard curve of BSA in the same buffer in a range of 2-0.005 mg/mL was used Samples ... (Jones et al 1993) In this work mg of pure protein were achieved out of a 400 mL culture, which highlights the advantages of recombinant expression and the fusion of an enzyme to a His-tag Previous...

Ngày tải lên: 21/06/2014, 02:20

... integrated elution peak areas, and the PDI’s oxidase and isomerase activities were expressed as the initial rate of decrease of linear tx3a and the initial rate of increase of nTx3.1, respectively To ... Oxidase and isomerase activities of cPDI measured using conotoxins as substrates Oxidasea,b Moles of reduced tx3a per mole of oxidase per (· 10)2) Isomeraseb,c Moles of nTx3.1 per mole of isomerase ... rate of decrease of the linear form, the original data were fitted by Y(t) ¼ e–kt · 100%, where Y is the percentage of the linear form, and t is the refolding time For the rate of increase of the...

Ngày tải lên: 07/03/2014, 05:20

... pathologies and because of the potential use of the toxins as biological weapons Alteration of their MHC and TCR binding capacity by site directed mutagenesis could be useful in the development of vaccines ... (genes kindly provided by U Utz and R P Sekaly, University of Montreal, Canada), were cloned between the NdeI and EcoRI restriction sites of the pET17b expression vector and expressed in E coli BL21(DE3) ... 30 and centrifuged for 10 at 12 000 g The supernatant was saved on ice and the pellet was resuspended in 50 mL of a : dilution of Tes and centrifuged as before Both supernatants were mixed and...

Ngày tải lên: 07/03/2014, 16:20

... probes As shown in Fig (A1 and A2) both probes gave specific hybridization bands of kb which are consistent with the sizes of each of the cDNAs The expression patterns of HRT1 and HRT2 mRNAs among the ... HRT2 The pETHRT1 and pETHRT2 were constructed and introduced into E coli BL21(DE3) The expression of these genes were induced by addition of 0.5 mM IPTG Overexpression of HRT1 and HRT2 is shown ... yielding the expression plasmids pETHRT1 and pETHRT2 Each of the expression plasmids was used for transformation of E coli BL21(DE3), and mL of an overnight culture of the transformant in Luria–Bertani...

Ngày tải lên: 07/03/2014, 21:20

... subtilis and B circulans should belong to subgroup Ib Expression and purification of AATB3 and its mutants To produce recombinant AATB3 and the three mutant proteins, the aatB3 gene and its mutants ... Purification and functional analysis of the recombinant wildtype (WT) and mutant AATB3 enzymes (A) Aliquots of purified enzyme for the wild-type and each AATB3 mutant were separated by SDS ⁄ PAGE and stained ... inserted into KpnI and EcoRI sites of pET30a(+) for the expression of protein AATB3; T7 promoter-based expression vector; Kmr The aatB3 fragment was inserted into KpnI and EcoRI sites of pUC19 for...

Ngày tải lên: 14/03/2014, 23:20

... proprothrombin), proFIX18 (residues )18 to )1 of proFactor IX), proFIX28 (residues )18 to +10 of proFactor IX), pro-e-TxIX/12 (residues )12 to )1 of e-TxIX precursor), e-TxIX12 (residues 1–12 of e-TxIX), ... of either the recombinant Conus carboxylase or the recombinant bovine carboxylase, and monitored carboxylation of FLEEL or e-TxIX12 as a function of propeptide concentration The effect of proFIX18 ... FLEEL, proPT28 and proFIX28, were Table Specific carboxylase and epoxidase activity of recombinant Conus carboxylase in the absence or presence of 400 lM proPT18 ND, not determined Expression system...

Ngày tải lên: 17/03/2014, 10:20

... sequence Expression and purification of the recombinant 2-5A synthetase from G cydonium Expression and purification of the porcine recombinant 2-5A synthetase N-terminally 6xHis-tagged construct The recombinant ... Experimental procedures of the porcine 2-5A synthetase (data not shown) Figure demonstrates the results of the purification of the recombinant proteins The occurrence of dominant bands of the recombinant ... evidence of a high degree of purification obtained by affinity chromatography Additionally, some fainter bands of higher and lower molecular weight could be seen in the preparations (Fig 1A) Bands of...

Ngày tải lên: 23/03/2014, 09:20

... solution and all run at the same time in the same solvent Standards of trehalose and maltose are applied to the sides of the paper to determine the locations of these sugars, and those areas of the ... by Q-TOF MS, and identified in the M smegmatis genome (shown in bold type and underlined) These peptides allowed the gene for TreS to be identified in the genome and its cloning and expression in ... formation and characterization of the products The conversion of trehalose to maltose was measured by determining the amount of reducing sugar resulting from the production of maltose The amount of...

Ngày tải lên: 23/03/2014, 13:20

... Exhaustive digestion of tamXG by Xeg74 created a set of XGOs from known amounts of substrate (0–1500 lg), and the approximate relationship between the glucose standard curve and the nmoles of XGOs produced ... of acetic acid, mL of p-anisaldehyde, mL of concentrated sulfuric acid) and then heated for h at 95 °C, as described by Chirco & Brown [19] and Jung et al [12] Glucose and xylose oligomer standards ... Pieces of the gels were rinsed with water, blotted on filter paper and chopped finely with a razor blade The dry mass of the material was approximately 1% of the blotted gel and overdigestion of GBX...

Ngày tải lên: 23/03/2014, 21:20

... lane 0, protein standards (C) Protein bands after affinity chromatography and renaturing process Lanes and 2, protein bands after separation by affinity column; lanes and 4, protein bands after renaturing ... immune system, the aberrant expression of cathelicidins is often associated with various disease processes [26,27] In the present study, the gene cloning and characterization of three avian cathelicidin ... Results Identification and characterization of quail cathelicidins Total RNA was extracted from the quail spleen On the basis of the end of the 5¢-UTR and the first 20 bp of the fowlicidin signal...

Ngày tải lên: 28/03/2014, 23:20

... fragments of m/z 58 (M+) and m/z 43 consistent with those of acetone standard Properties of NaoA The pI of NaoA is about 5.2 according to the standard plot between the protein’s pI and its migration ... reading and help in preparation of this paper REFERENCES Venulet, J & Van-Etten, R.L (1970) Biochemistry and pharmacology of the nitro and nitroso group In The Chemistry of the Nitro and Nitroso ... formation of nitrite using the method of Ida et al [31] Km and Vmax of purified NaoA were determined from a double-reciprocal plot according to the LineweaverBurk equation [32] RESULTS Cloning of 1.5-kb...

Ngày tải lên: 31/03/2014, 08:20

... Lane 4, DNA marker 2000 Page of 11 Expression and purification of the gE recombinant protein To obtain a highly expressed level of pET32a/DPV-gE protein, the recombinant expression vectors pET32a/ ... (EcoRI and XhoI) Escherichia coli BL21(pLysS), BL21(DE3) and Rosseta cells were individually transformed with the positive recombinant plasmid and used for protein expression Expression and Purification ... Fund of the Key Scientific and Technical Innovation Project, Ministry of Education of China (706050), and the Cultivation Fund of the Key Scientific and Technical Innovation Project, department of...

Ngày tải lên: 12/08/2014, 04:20

... Cytoplamic protein (CP) 64 3.6 Expression and purification of recombinant HSP20 (rHSP20) 64 3.6.1 Induced expression of recombinant protein (rHSP20) 64 3.6.2 Purification of rHSP20 by Affinity chromatography ... patients’ samples; Prof Douglas E Berg of Washington University School of Medicine, USA; Prof B Marshall of University of Western Australia, Australia and A/Prof N Aoyama of Kobe University, Japan ... residues of HSP20 RESULTS 4.1 Preparation of recombinant HSP20 (rHSP20) 85 4.1.1 Construction of rHSP20 expression vector 85 4.1.2 Expression and purification rHSP20 protein 87 4.2 Preparation and...

Ngày tải lên: 12/09/2015, 10:19

... Loss of Osh7p function increases sterol esterification 107 4.5 Understanding of the nature of the interaction between Osh7p and Vps4p and future directions 108 4.6 Understanding the nature of Osh7p ... structure of Vps4p 38 Model for ATP-driven cycle of Vps4p in vivo and in vitro 39 Schematic figure of the protein structure of Osh7p 40 10 Outline of short flanking homology strategy for disruption of ... 1993) Thus, the regulation of sterol homeostasis appears to require many of the similar genes and proteins in yeast and human and much of the work in defining the role of sterol in eukaryotic membranes...

Ngày tải lên: 03/10/2015, 20:57

... D4-3-ketosteroids, but have been shown to reduce both 3- and 20-ketosteroids Thus, DpARs are ketosteroid reductases instead of D4-3-ketosteroid 5b -reductases As inferred from the results of their ... of reduction of such substrates; they not react with NADP or NAD As shown in Table 1, both enzymes can metabolize aldose and ketose substrates, as well as steroids, in the presence of NADH cofactor ... 2002 Aldo- keto reductases in D purpurea leaves (Eur J Biochem 269) 2845 Fig Alignment of the amino-acid sequences of proteins within a closely related AKR family Sequences aligned are DpAR1 and...

Ngày tải lên: 18/03/2014, 01:20

... Bacterial expression and purification of recombinant zebrafish cytosolic STs To amplify the two zebrafish ST cDNAs for subcloning into the prokaryotic expression vector pGEX-2TK, two sets of sense and ... Detection of zebrafish SULT1 ST1 and ST2 mRNAs and (B) Western blot analysis of zebrafish SULT1 ST1 protein (A) Detection of zebrafish SULT1 ST1 and ST2 mRNAs in cultured zebrafish cells (lanes and 3) and ... dose-dependence of the regulation of the activity of the zebrafish ST by these divalent metal cations and their modes of action Fig Effects of divalent metal cations on the sulfating activity of the zebrafish...

Ngày tải lên: 08/03/2014, 02:20

... lg of platelet protein and 0.5 lg of GST and GST±Rab proteins; (B) an [a-32P]GTP overlay [17] of a nitrocellulose blot of a gel containing 40 lg of platelet protein, 0.5 lg of GST and 0.5 lg of ... form of Rab32 (see Discussion) Fig Puri®cation and properties of wild-type and mutant Rab31 and Rab32 expressed as GST±fusion proteins Samples of platelet particulate fraction protein and of puri®ed ... cycles) and at 72 °C, for a total of 21 cycles The PCR product was puri®ed using a QIAquickTM kit and cloned into the BamHI and XhoI sites of the pGEX-4T-1 expression vector The sequence of the...

Ngày tải lên: 08/03/2014, 16:20

... isolation of a bovine tryptase gene and cDNA encoding a new tryptase isoform (BLT), which is in turn the only one isoform expressed in bovine lung Furthermore, analysis of BLCT and BLT expression ... tryptase isoform (BLT) was obtained from lung mRNA by RT-PCR, using primers N9 and N10, and by subsequent cloning and sequencing Based on this partial sequence, 5¢ RACE experiments and RT-PCR ... fraction contains 0.3 nmol of active Ó FEBS 2003 Tissue-specific expression of bovine tryptases (Eur J Biochem 270) 513 Fig Comparison of promoter sequences and 5¢-UTRs in BLT and BLCT genes Identical...

Ngày tải lên: 17/03/2014, 09:20