cloning expression and purification of recombinant proteins

... Gami bla Figure strategy for expression of LASV proteins GP1, GP2, and NP in E coli using pMAL vectors Cloning Cloning strategy for expression of LASV proteins GP1, GP2, and NP in E coli using pMAL ... 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 LASV proteins ... 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 ... Collection (ATCC 14468) The E coli strains DH5a and HMS-F [25] were used for cloning and expression studies, respectively HMS-F is a derivative of the expression strain HMS174(DE-3) (Novagen) HMS174(DE-3)...

Ngày tải lên: 17/03/2014, 10:20

... complex and assembled into a particulate structure in the absence of genomic RNA Figure Assembly of VLPs by expression of RVFV N and Gc proteins Assembly of VLPs by expression of RVFV N and Gc proteins ... http://www.virologyj.com/content/5/1/82 Figure Expression of N, Gn and Gc proteins produced virus-like particles Expression of N, Gn and Gc proteins produced virus-like particles A) Sf9 cells were infected with the recombinant ... purification of proteins and VLPs, and EM studies CC carried out cell surface expression and cell to cell fusion studies PR contributed in the coordination and design of the study and helped in...

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 ... (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

... for purification of two Histagged recombinant proteins, His-ProT and His-Mre11, overexpressed in Escherichia coli Results Relative expression of His-tagged recombinant proteins Recombinant proteins ... magnetic beads and non-specific protease Protein Expression and Purification 2002, 25:426-429 Safarik I, Safarikova M: Magnetic techniques for the isolation and purification of proteins and peptides ... fractions of His -recombinant proteins bound onto the different concentrations of three Nickel-coated magnetic matrices His-ProT and His-Mre11 recombinant proteins in soluble cell extract (SCE) of E.coli...

Ngày tải lên: 11/08/2014, 00:23

... 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

... 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 proteins ... 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 ... characterized by means of a recombinant protein technique Results Expression and purification of His-tagged proteins N- and C-terminally hexahistidine tagged constructs of the 2-5A synthetase...

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 ... 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

... 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

... 25 1.3.1 Structure of Osh proteins 25 1.3.2 Localization of Osh proteins 27 1.3.3 Function of Osh proteins 27 1.3.3.1 Role of OSH in maintaining sterol-lipid distribution and vacuolar integrity ... 70 2.16.1 Purification of recombinant GST-Osh7p and GST proteins 70 2.16.1.1 Preparation of the bacterial lysate 70 2.16.1.2 Affinity chromatography to purify the GST-Osh7p and GST proteins 70 ... 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...

Ngày tải lên: 03/10/2015, 20:57

... 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 contain ... 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 ... 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

... 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