cloning expression and purification of chimeric polyprotein constructs

... 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 ... 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 ... Lysozyme, Micrococcus lysodeikticus and RNase A were products of Sigma (St Louis, MO, USA) Other reagents were of analytical grade Gene cloning of cPDI The full-length cDNA of cPDI was amplified by RT-PCR...

Ngày tải lên: 07/03/2014, 05:20

... hVb2.1hCb2 and hVb1hCb2 (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 ... 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 ... 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 ... 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 ... into KpnI and EcoRI sites of pET30a(+) for the expression of protein D232N; T7 promoter-based expression vector; Kmr The fragment from pUCK270H was inserted into KpnI and EcoRI sites of pET30a(+)...

Ngày tải lên: 14/03/2014, 23:20

... 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

... 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 ... trypsinogen, pI 9.3 The pI of NaoA was determined from a standard curve of pI and migration distance (cm) of protein standards Enzyme assay and analytical methods E coli BL21(DE3) containing plasmid...

Ngày tải lên: 31/03/2014, 08:20

... 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

... protein One unit is defined 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 ... 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 ... though separation of the distinct activities was tried by purification, the presence of impurities could not be excluded Therefore, reproducible and reliable methods for separation and purification...

Ngày tải lên: 21/06/2014, 02:20

... 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 ... analysis of the DPV gE protein Materials and methods Materials DPV CHv strains and the rabbit anti-DPV were provided by Key Laboratory of Animal Disease and Human Health of Sichuan Province The expression ... (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...

Ngày tải lên: 12/08/2014, 04:20

... 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 ... caring, encouragement and understanding i Table of contents Table of contents Content Page ACKNOWLEDGEMENT i LIST OF FIGURES ii LIST OF TABLES v LIST OF ABBREVIATIONS vii LIST OF PUBLICATIONS xi ... 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...

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

... shown in Fig 7b, the level of cHSPA6 expression reached at its maximal level within 1h of induction and incubation at 37 oC The level of cHSPA6 remained unchanged up to 24h of post-induction incubation ... allows very sensitive detection (1–10 ng of protein per band) with negligible background staining 2.1 Results Expression of recombinant cHSPA6 The result express of recombinant cHSPA6 vector which ... were induced with varying concentrations of IPTG (0, 10, 25, 50, 100, 250, 500 and 1000 μM) and further expressions were made for h at 37 °C An equal amount of soluble protein extract was analyzed...

Ngày tải lên: 24/10/2016, 14:24

... 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 ... 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...

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

... of the polyprotein expressed in prokaryotic systems, or due to low levels of expression in transfected mammalian cells The baculovirus system offered the advantage of high expression levels and ... protein purification, western blots and analysis of the MALDI-TOF data; MC carried out cloning of HEV ORF1 All authors read and approved the final manuscript Acknowledgements We thank Dr S.K Panda ... infected T ni cells (Fig 2A and 2B, lane 6) The expression of the ~192 kDa fragments and accumulation of smaller fragments as a function of time was indic- Effect of cysteine protease inhibition...

Ngày tải lên: 20/06/2014, 01:20

... in order to secrete IL-1b and TNF-a Hypoxia ⁄ SD induces the transcription and secretion of IL-10 Because of the lack of secretion of the inflammatory cytokines IL-1b and TNF-a from hypoxia ⁄ SD-stimulated ... presence and absence of IL-10 *P < 0.05 and **P < 0.01 versus Cont group (D) Representative western blots for collagen I and III in the presence and absence of 0.1 lM angiotensin II and IL-10 ... effects of MSCs in vitro mediator of the cells’ paracrine anti-fibrotic effects These findings help to improve our understanding of the cellular and molecular basis of MSCs’ anti-inflammatory and paracrine...

Ngày tải lên: 18/02/2014, 04:20