cloning expression and purification of idps in pfs 2

... activity increased proportionally BuOH extract 26 70 ± 22 5 28 20 ± 25 3 321 0 ± 24 4 Toluene/hexane extract 28 0 ± 68 28 0 ± 45 320 ± 65 28 10 3150 3700 5190 ± ± ± ± 29 8 173 340 414 25 10 22 30 27 70 7 120 ± ... Biochem 27 0) Fig Overexpression of HRT1 and HRT2 in E coli and purification of HRT2 The pETHRT1 and pETHRT2 were constructed and introduced into E coli BL21(DE3) The expression of these genes were induced ... AAM 928 90), AAM 928 81, AAM 928 79, BAB 920 23 (AAM 928 83, AAM 928 84, AAM 928 85, AAM 928 87, AAM 928 88), BAB 920 24 and AAM 928 82 (AAM 928 86) (submitted to GenbankTM and DDBJ by Coldren et al and Sando et al respectively)...

Ngày tải lên: 07/03/2014, 21:20

... Structure of oxysterols 17 1 .2. 2 Roles of oxysterols 19 1 .2. 3 Oxysterol-binding protein 22 Yeast OSBP homologues 25 1.3.1 Structure of Osh proteins 25 1.3 .2 Localization of Osh proteins 27 1.3.3 ... competent cells and transformation 50 2. 5.1.8 Selection and DNA sequencing 51 2. 5 .2 Other subclonings 52 2.5 .2. 1 Generation of pJG4-5-OSH6cc construct 52 2.5 .2. 2 Generation of YEplac-OSH7-GFP and YCplac-OSH7-GFP ... plasmids 2. 6 52 2.5 .2. 3 Generation of pADNS-OSH7 construct 52 2.5 .2. 4 Generation of pGEX-4T -2- OSH7 construct 52 Deletion of OSH7 53 2. 6.1 PCR amplification and purification 53 2. 6 .2 Transformation of...

Ngày tải lên: 03/10/2015, 20:57

... within 1h of induction and incubation at 37 oC The level of cHSPA6 remained unchanged up to 24 h of post-induction incubation at 37 oC, indicating that the camel HSPA6 is well folded, soluble and ... uninduced in 2 LB; 7, induced in 2 LB; 8, uninduced in TB; 9, induced in TB 2. 2.3 Inducer concentration optimization A higher IPTG concentration has no significant effect on the yield of cHSPA6 ... pre-induction growth on the final cell density IPTG (μM) 25 25 25 25 2. 2.5 Cell d 0.3 0.6 1 .2 1.8 Post-induction incubation optimization As shown in Fig 7b, the level of cHSPA6 expression reached at...

Ngày tải lên: 24/10/2016, 14:24

... LW (20 04) A conserved arginine plays a role in the catalytic cycle of the protein disulphide isomerases J Mol Biol 335, 28 3– 29 5 22 Lambert N & Freedman RB (1983) Kinetics and specificity of homogeneous ... protein has a calculated molecular mass of 54 913.7 Da and an isoelectric point of 4.6 The cPDI contains four thioredoxin domains and an acidic C-terminal tail (a, b, b¢, a¢ and c) Two thioredoxin ... 7.5) containing mm GSH, 30 lm insulin, 120 lm NADPH, 0.5 units of glutathione reductase, mm EDTA, and 0.7 lm PDI The assay mixture (without insulin and PDI) was equilibrated at 25 °C, and the NADPH...

Ngày tải lên: 07/03/2014, 05:20

... kanamycin resistant expression vector pET26b and expressed as inclusion bodies [ 12] in Escherichia coli BL21(DE3) (Stratagene, La Jolla, CA) Two other b chains, hVb2.1hCb2 and hVb1hCb2 (genes kindly ... the TCR binding site of SEB and SEC3 [ 12, 13,19 ,21 ], showed that Cys26 is not only exposed in the protein surface (Fig 2) , but would be in the putative TCR binding site Consequently, a point mutation ... the increased interest in these molecules in the treatment of several pathologies and because of the potential use of the toxins as biological weapons Alteration of their MHC and TCR binding...

Ngày tải lên: 07/03/2014, 16:20

... amino acids of the glutamate and aspartate families, alanine, and polyamines In Bacillus subtilis and its Closest Relatives 1356 22 23 24 25 26 27 28 29 30 31 32 (Sonenshein AL ed), pp 20 3 23 1 ... AAT and PLP, the Asp2 32 and Lys270 residues (corresponding to Asp 222 and Lys258 in E coli AAT) were replaced with Asn and His using site-directed mutagenesis to obtain the mutants D232N and K270H, ... pUCD232N was inserted 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...

Ngày tải lên: 14/03/2014, 23:20

... crab Glucose (ug/mL hemolymph) A 300 ** 20 0 100 22 to 22 22 to 29 22 to Temperature (°C) Lactate (ug/ mL hemolymph) B 180 *** 120 ** 60 * 22 to 22 22 to 29 22 to Temperature (°C) 1e+9 1e+8 b 1e+7 ... higher, at 29 °C In contrast to the results obtained with hypoxia and emersion, as shown in Fig 7, there was little change in AK and eIF4A expression in the ES and PO at 22 °C and 29 °C, except ... varicosities in trunks of the PO Cloning and sequencing of PO-CHH cDNA The first PCR of PO cDNA with a combination of a set of primers LF1 and LR1 (Table 1) produced an amplicon of 500 bp On the basis of...

Ngày tải lên: 16/03/2014, 06:20

... protects cells and cellular proteins from damage by oxygen radicals J Biol Chem 27 6, 24 261 24 267 Singer, M.A & Lindquist, S (1998) Multiple effects on protein folding in vitro and in vivo Mol Biol ... electroporated into the E coli strain HMS-F [29 ] The resulting ampicillin- and tetracycline-resistant transformant was cultured with and without IPTG, and expression of a (His)6-tagged protein of the ... by cutting a strip of the paper into 0.5 cm pieces, from the origin to the solvent front, and counting each strip in a scintillation counter RESULTS Cloning and expression of M tuberculosis TPS...

Ngày tải lên: 17/03/2014, 10:20

... smegmatis mc2155 was provided by W R Jacobs Jr., Albert Einstein College of Medicine, New York The E coli strains TOP10 and BL21Star (DE3) (Invitrogen) were used for cloning and expression studies, ... Abraham, E.C.,Carroll, J.D & Elbein, A.D (20 03) Purification, Ó FEBS 20 04 Interconversion of trehalose and maltose (Eur J Biochem 27 1) 426 9 cloning, expression and properties of mycobacterial trehalose– ... (NH4)2SO4 Gel filtration DE- 52 Hydroxyapatite Aminohexylagarose Phenylsepharose 11448 4040 1 720 120 42 65 25 0 31 416 18 748 9168 7804 5.7 7.9 10.9 76.4 185 1.4 2. 0 13 33 100 49 29 14 12 1 .2 525 0...

Ngày tải lên: 23/03/2014, 13:20

... Doner, L.W & Irwin, P.L (19 92) Assay of reducing end-groups in oligosaccharide homologues with 2, 2¢ bicinchoninate Anal Biochem 20 2, 50–53 18 Pauly, M., Andersen, L.N., Kauppinen, S., Kofod, L.V., ... reducing end of XG, releasing GG, XG, or LG [33] This protein is unique in having four regions of amino acids (23 5 25 1; 310–318; 361–3 72; 398–413) that are not found in the other family-74 proteins ... [11 ,22 ] using this signal sequence and it has a convenient NotI site at its C terminus, which allows cloning in- frame with alanine as the first amino acid of the mature protein An alignment of...

Ngày tải lên: 23/03/2014, 21:20

... 0.3 0 .2 0.4 1.0 0.79 8.05 1.3 20 .2 > 26 .9 20 .2 20 .2 20 .2 2.5 2. 5 1.3 20 .2 > 26 .9 20 .2 20 .2 20 .2 2.5 2. 5 0.7 2. 8 2. 8 2. 8 2. 8 2. 8 ND 0.7 0.7 2. 8 2. 8 2. 8 2. 8 2. 8 ND 0.7 4 .2 ND > 22 .3 > 22 .3 > 22 .3 ... RLL 62 62 62 62 62 62 62 62 62 72 72 72 72 73 72 72 73 72 73 70 72 72 68 Pc-CATH1 Pc-CATH2 Pc-CATH3 Cc-CATH1 Cc-CATH2 Cc-CATH3 Fowlicidin1 Fowlicidin2 Fowlicidin3 Ea CATH1 Ec CATH1 Ec CATH2 Ec ... G C C 122 122 122 122 122 122 122 122 122 130 130 130 130 133 130 143 131 130 131 139 134 134 143 Pc-CATH1 Pc-CATH2 Pc-CATH3 Cc-CATH1 Cc-CATH2 Cc-CATH3 Fowlicidin1 Fowlicidin2 Fowlicidin3 Ea...

Ngày tải lên: 28/03/2014, 23:20

... b-lactoglobulin A, pI 5.1; myoglobin, pI 6.8/7 .2; 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 ... enzyme of Streptomyces (Eur J Biochem 26 9) 6305 Fig SDS/PAGE of NaoA expressed in E coli and its purification Lane 1, total protein from BL21(DE3)/pET23b; lane 2, total protein from strain BL21(DE3)/pNA101; ... examined (Table 2) Mn2+ increased the enzyme activity slightly, and Cu2+ inhibited it Mg2+ and Ca2+ did not affect NaoA activity Thiol groups may be involved in the active site of NaoA because...

Ngày tải lên: 31/03/2014, 08:20

... Dr Randal Schoepp kindly provided the following LASV-specific mAbs: NP-specific mAbs 522 73-8 and L2-54-6A; GP1-specific mAb L 52- 74-7A; and GP2-specific mAbs L 52- 2 72- 7, L 52- 121 -22 , and L 52- 2 727 , ... GP1-MBP are indicated 98 62 49 38 28 17 14 GP2-MBP MBP GP2 Figure transformed with construct from E coli Rosetta gami cellsand purification of LASV GP2pMAL-c2x:GP2 Expression Expression and purification ... 30°C, and 37°C to an A600 LASV Gene Expression System pMAL-p2x:GP1 pMAL-p2x:GP2 pMAL-p2x:NP pMAL-c2x:GP1 pMAL-c2x:GP2 pMAL-c2x:NP GP1 GP2 NP GP1 GP2 NP Rosetta 2( DE3) Rosetta 2( DE3) Rosetta 2( DE3)...

Ngày tải lên: 20/06/2014, 01:20

... Vector 2, 5-DKCMO_pET -28 for expression of recombinant 2, 5-DKCMO from P putida NCIMB 10007 under control of T7 promoter in E coli BL21 The 2, 5-DKCMO-gene 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 were measured in triplicates in three different ... afterwards ligated into the expression vector pET -28 b fused to the N-terminal His-tag to allow a functional expression of the 2, 5-DKCMO in E coli and easy protein purification by affinity chromatography...

Ngày tải lên: 21/06/2014, 02:20

... Damme J: Selective induction of CCL18/PARC by staphylococcal enterotoxins in mononuclear cells and 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 enhanced levels in septic and rheumatoid ... secretion of macrophage inflammatory protein and interleukin by human neutrophils: a possible mechanism of neutrophil recruitment to sites of inflammation in synovitis J Exp Med 1995, 1 82: 2019 -20 25 ... volume of 25 μl at 37°C for 60 minutes and at 93°C for minutes PCR was performed in a 25 μl reaction mixture containing μl of cDNA sample, 0. 625 U of Taq polymerase (Qiagen), 0 .2 mM dNTPs (Invitrogen)...

Ngày tải lên: 09/08/2014, 10:21

... degeneration Spine 20 02, 27 : 127 8- 128 5 Jackson HC 2nd, Winkelmann RK, Bickel WH: Nerve endings in the human spinal column and related structures J Bone Joint Surg Am 1966, 48: 127 2- 128 1 Bogduk N, ... expression and innervation of the painful intervertebral disc J Pathol 20 02, 197 :28 6 -29 2 Freemont AJ, Peacock TE, Goupille P, Hoyland JA, O'Brien J, Jayson MIV: Nerve ingrowth into diseased intervertebral ... fully examined A role for the proinflammatory cytokines TNFα and IL-1β in innervation of the IVD [22 ] and in degeneration of the IVD has been demonstrated by a number of authors [23 -25 ] Additionally...

Ngày tải lên: 09/08/2014, 11:20

... http://www.virologyj.com/content/7/1/ 120 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 cloning and identification of viral nucleocapsid protein gene High Technol Lett 20 06, 16:948-953 Bradford MM: A rapid and ... expressed level of the pET32a/DPV-gE protein was better in Rosseta than in BL21(DE3) host strain, but the recombinant protein was not expressed in BL21(pLysS) (Fig 2A) And the expression level of the ... localization of the gE protein using the rabbit polyclonal antiserum specific to it in DPV-infected cells We examined the expression of gE protein in DPV-infected cells using Western blotting, and analyzed...

Ngày tải lên: 12/08/2014, 04:20

... established in resting memory CD4+ T-cells in vitro following incubation with the chemokines CCL19 and CCL21 (ligands for CCR7), CXCL9 and CXCL10 (ligands for CXCR3) and CCL20 (ligand for CCR6) [11, 12] ... block in initiation and completion of HIV transcription as well as a block in translation of viral proteins by the expression of microRNAs (reviewed in [21 ]] In addition, a clear block in export of ... using anti-CD3 and anti-CD28 [1, 2] ; or other stimuli such as IL-7 [23 ], IL2 [23 ], the protein kinase C (PKC) activator prostratin [24 , 25 ], histone deacetylase inhibitors (HDACi) such as vorinostat...

Ngày tải lên: 13/08/2014, 01:21

... 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 65 3.7 Raising ... 41 3 .2. 3 Mini-preparation of plasmid DNA 43 3 .2. 4 Construction of recombinant HSP20 expression vector 44 3 .2. 5 Construction of hsp20::aphA gene-targeting vector 46 3 .2. 6 Transformation of H pylori ... comparison of substitutions at 14th – 16th amino acid 84 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...

Ngày tải lên: 12/09/2015, 10:19

... to that of the cell cycle inhibitor p27kip1 p27kip1 was shown to be targeted by miR -22 1 and miR -22 2 at two sites on its 19 3’UTR Over -expression of miR -22 1 and miR -22 2 in both types of tumors ... progression, including cyclin E and cyclin A (Arroyo and Raychaudhuri, 19 92) Cyclin E and cyclin A complex with CDK2 to promote G1 and S phase progression Cyclin B interacts with CDK1 during late G2 and ... Screening of predicted targets of miR -21 0 59 4.5 Screening of predicted targets of miR-193a 63 Discussion 67 5.1 Differential expression of miRNAs during cell cycle phases 67 5 .2 Role of miR -21 0 in...

Ngày tải lên: 05/10/2015, 22:31