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81.
We report an efficient system to produce and display properly folded disulfide-rich proteins facilitated by coupled complementary DNA (cDNA) display and protein disulfide isomerase-assisted folding. The results show that a neurotoxin protein containing four disulfide linkages can be displayed in the folded state. Furthermore, it can be refolded on a solid support that binds efficiently to its natural acetylcholine receptor. Probing the efficiency of the display proteins prepared by these methods provided up to 8-fold higher enrichment by the selective enrichment method compared with cDNA display alone, more than 10-fold higher binding to its receptor by the binding assays, and more than 10-fold higher affinities by affinity measurements. Cotranslational folding was found to have better efficiency than posttranslational refolding between the two investigated methods. We discuss the utilities of efficient display of such proteins in the preparation of superior quality proteins and protein libraries for directed evolution leading to ligand discovery. 相似文献
82.
G. Mattson E. Conklin S. Desai G. Nielander M. D. Savage S. Morgensen 《Molecular biology reports》1993,17(3):167-183
The various aspects of chemical crosslinking are addressed. Crosslinker reactivity, specificity, spacer arm length and solubility characteristics are detailed. Considerations for choosing one of these crosslinkers for a particular application are given as well as reaction conditions and practical tips for use of each category of crosslinkers.Abbreviations ABH
azidobenzoyl hydrazide
- ANB- NOS
N-5-azido-2-nitrobenzoyloxysuccinimide
- ASIB
1-(p-azidosalicylamido)-4-(iodoacetamido)butane
- ASBA
4-(p-azidosalicylamido)butylamine
- APDP
N-[4-(p-azidosalicylamido) butyl]-3(2-pyridyldithio)propionamide
- APG
p-azidophenyl glyoxal monohydrate
- BASED
bis-[-(4-azidosalicylamido)ethyl] disulfide
- BMH
bismaleimidohexane
- BS3
bis(sulfosuccinimidyl) suberate
- BSOCOES
bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone
- DCC
N,N-dicyclohexylcarbodiimide
- DFDNB
1,5-difluoro-2,4-dinitrobenzene
- DMA
dimethyl adipimidate·2HCl
- DMP
dimethyl pimelimidate·2HCl
- DMS
dimethyl suberimidate·2HCl
- DPDPB
1,4-di-(3,2-pyridyldithio)propionamido butane
- DMF
dimethylformamide
- DMSO
dimethylsulfoxide
- DSG
disuccinimidyl glutarate
- DSP
dithiobis(succinimidylpropionate)
- DSS
disuccinimidyl suberate
- DST
disuccinimidyl tartarate
- DTSSP
3,3-dithiobis (sulfosuccinimidylpropionate)
- DTBP
dimethyl 3,3-dithiobispropionimidate·2HCl
- EDC or EDAC
1-ethyl-3-(3-dimethylaminopropyl)carbodimide hydrochloride
- EDTA
ethylenediaminetetraacetic acid disodium salt, dihydrate
- EGS
ethylene glycolbis(succinimidylsuccinate)
- GMBS
N--maleimidobutyryloxysuccinimide ester
- HSAB
N-hydroxysuccinimidyl-4-azidobenzoate
- HEPES
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
- MBS
m-maleimidobenzoyl-N-hydroxysuccinimide ester
- MES
4-morpholineethanesulfonic acid
- NHS
N-hydroxysuccinimide
- NHS-ASA
N-hydroxysuccinimidyl-4-azidosalicylic acid
- PMFS
phenylmethylsulfonyl fluoride
- PNP-DTP
p-nitrophenyl-2-diazo-3,3,3-trifluoropropionate
- SAED
sulfosuccinimidyl 2-(7-azido-4-methylcoumarin-3-acetamide) ethyl-1,3-dithiopropionate
- SADP
N-succinimdyl (4-azidophenyl)1,3-dithiopropionate
- SAND
sulfosuccinimidyl 2-(m-azido-o-nitrobenzamido)-ethyl-1,3-dithiopropionate
- SANPAH
N-succinimidyl-6(4-azido-2-nitrophenyl-amino)hexanoate
- SASD
sulfosuccinimidyl 2-(p-azidosalicylamido)ethyl-1,3-dithiopropionate
- SATA
N-succinimidyl-S-acetylthioacetate
- SDBP
N-hydroxysuccinimidyl-2,3-dibromopropionate
- SIAB
N-succinimidyl(4-iodoacetyl)aminobenzoate
- SMCC
succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate
- SMPB
succinimidyl 4-(p-maleimidophenyl) butyrate
- SMPT
4-succinimidyloxycarbonyl--methyl--(2-pyridyldithio)-toluene
- sulfo-BSOCOES
bis[2-sulfosuccinimidooxycarbonyloxy) ethyl]sulfone
- sulfo-DST
disulfosuccinimidyl tartarate
- sulfo-EGS
ethylene glycolbis(sulfosuccinimidylsuccinate)
- sulfo-GMBS
N--maleimidobutyryloxysulfosuccinimide ester
- sulfo-MBS
m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester
- sulfo-SADP
sulfosuccinimidyl(4-azidophenyldithio)propionate
- sulfo-SAMCA
sulfosuccinimidyl 7-azido-4-methylcoumarin-3-acetate
- sulfo-SANPAH
sulfosuccinimidyl 6-(4-azido-2-nitrophenylamino)hexanoate
- sulfo-SIAB
sulfosuccinimidyl(4-iodoacetyl)aminobenzoate
- sulfo-SMPB
sulfo-succinimidyl 4-(p-maleimidophenyl)butyrate
- sulfo-SMCC
sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate
- SPDP
N-succinimidyl 3-(2-pyridyldithio)propionate 相似文献
83.
Sevier CS Kadokura H Tam VC Beckwith J Fass D Kaiser CA 《Protein science : a publication of the Protein Society》2005,14(6):1630-1642
Three different classes of thiol-oxidoreductases that facilitate the formation of protein disulfide bonds have been identified. They are the Ero1 and SOX/ALR family members in eukaryotic cells, and the DsbB family members in prokaryotic cells. These enzymes transfer oxidizing potential to the proteins PDI or DsbA, which are responsible for directly introducing disulfide bonds into substrate proteins during oxidative protein folding in eukaryotes and prokaryotes, respectively. A comparison of the recent X-ray crystal structure of Ero1 with the previously solved structure of the SOX/ALR family member Erv2 reveals that, despite a lack of primary sequence homology between Ero1 and Erv2, the core catalytic domains of these two proteins share a remarkable structural similarity. Our search of the DsbB protein sequence for features found in the Ero1 and Erv2 structures leads us to propose that, in a fascinating example of structural convergence, the catalytic core of this integral membrane protein may resemble the soluble catalytic domain of Ero1 and Erv2. Our analysis of DsbB also identified two new groups of DsbB proteins that, based on sequence homology, may also possess a catalytic core similar in structure to the catalytic domains of Ero1 and Erv2. 相似文献
84.
Flexible Perovskite Photovoltaic Modules and Solar Cells Based on Atomic Layer Deposited Compact Layers and UV‐Irradiated TiO2 Scaffolds on Plastic Substrates 下载免费PDF全文
85.
Regulation of matrix remodelling phenotype in gingival fibroblasts by substratum topography 下载免费PDF全文
Shawna S. Kim Weiyan Wen Paul Prowse Douglas W. Hamilton 《Journal of cellular and molecular medicine》2015,19(6):1183-1196
Gingival connective tissue often has a composition resembling that of scar surrounding dental implant abutments. Increased cell adhesion, α‐smooth muscle actin (α‐SMA) expression and increased extracellular matrix deposition are a hallmark of fibrotic cells, but how topographic features influence gingival fibroblast adhesion and adoption of the α‐SMA positive myofibroblast phenotype associated with scarring is unknown. The purpose of the present study was to demonstrate whether implant topographies that limit adhesion formation would reduce myofibroblast differentiation and extracellular matrix deposition. Human gingival fibroblasts were cultured on PT (smooth) and SLA (roughened) titanium discs for varying time‐points. At 1 and 2 weeks after seeding, incorporation of α‐SMA into stress‐fibre bundles and fibronectin deposition was significantly higher on PT than SLA surfaces indicating differentiation of the cells towards a myofibroblast phenotype. Analysis of adhesion formation demonstrated that cells formed larger adhesions and more stable adhesions on PT, with more nascent adhesions observed on SLA. Gene expression analysis identified up‐regulation of 15 genes at 24 hrs on SLA versus PT associated with matrix remodelling. Pharmacological inhibition of Src/FAK signalling in gingival fibroblasts on PT reduced fibronectin deposition and CCN2 expression. We conclude that topographical features that reduce focal adhesion stability could be applied to inhibit myofibroblast differentiation in gingival fibroblasts. 相似文献
86.
Prions are the etiological agent of fatal neurodegenerative diseases called prion diseases or transmissible spongiform encephalopathies. These maladies can be sporadic, genetic or infectious disorders. Prions are due to post-translational modifications of the cellular prion protein leading to the formation of a β-sheet enriched conformer with altered biochemical properties. The molecular events causing prion formation in sporadic prion diseases are still elusive. Recently, we published a research elucidating the contribution of major structural determinants and environmental factors in prion protein folding and stability. Our study highlighted the crucial role of octarepeats in stabilizing prion protein; the presence of a highly enthalpically stable intermediate state in prion-susceptible species; and the role of disulfide bridge in preserving native fold thus avoiding the misfolding to a β-sheet enriched isoform. Taking advantage from these findings, in this work we present new insights into structural determinants of prion protein folding and stability. 相似文献
87.
Amyotrophic Lateral Sclerosis is a motor neurodegenerative disease which is characterized by progressive loss of motor neurons followed by paralysis and eventually death. In human, VAMP-associated protein B (VAPB) is the causative gene of the familial form of ALS8. Previous studies have shown that P56S and T46I point mutations of hVAPB are present in this form of ALS. Recently, another mutation, V234I of hVAPB was found in one familial case of ALS. This is the first study where we have shown that V234I-VAPB does not form aggregate like other two mutants of VAPB and localizes differently than the wild type VAPB. It induces Ubiquitin aggregation followed by cell death. We propose that V234I-VAPB exhibits the characteristics of ALS in spite of not having the typical aggregation property of different mutations in various neurodegenerative diseases. 相似文献
88.
89.
C.C. García M. Djavani E.B. Damonte 《Biochemical and biophysical research communications》2010,393(4):625-169
The promyelocytic leukemia protein (PML) forms nuclear bodies (NB) that can be redistributed by virus infection. In particular, lymphocytic choriomeningitis virus (LCMV) influences disruption of PML NB through the interaction of PML with the arenaviral Z protein. In a previous report, we have shown that the disulfide compound NSC20625 has antiviral and virucidal properties against arenaviruses, inducing unfolding and oligomerization of Z without affecting cellular RING-containing proteins such as the PML. Here, we further studied the effect of the zinc-finger-reactive disulfide NSC20625 on PML-Z interaction. In HepG2 cells infected with LCMV or transiently transfected with Z protein constructs, treatment with NSC20625 restored PML distribution from a diffuse-cytoplasmic pattern to punctate, discrete NB which appeared identical to NB found in control, uninfected cells. Similar results were obtained in cells transfected with a construct expressing a Z mutant in zinc-binding site 2 of the RING domain, confirming that this Z-PML interaction requires the integrity of only one zinc-binding site. Altogether, these results show that the compound NSC20625 suppressed Z-mediated PML NB disruption and may be used as a tool for designing novel antiviral strategies against arenavirus infection. 相似文献
90.
Rituraj Pal Karina Schnittker 《Biochemical and biophysical research communications》2010,392(4):567-571
Protein disulfide isomerase (PDI), the chief endoplasmic reticulum (ER) resident oxidoreductase chaperone that catalyzes maturation of disulfide-bond-containing proteins is involved in the pathogenesis of both Parkinson’s (PD) and Alzheimer’s (AD) diseases. S-nitrosylation of PDI cysteines due to nitrosative stress is associated with cytosolic debris accumulation and Lewy-body aggregates in PD and AD brains. We demonstrate that the polyphenolic phytochemicals curcumin and masoprocol can rescue PDI from becoming S-nitrosylated and maintain its catalytic function under conditions mimicking nitrosative stress by forming stable NOx adducts. Furthermore, both polyphenols intervene to prevent the formation of PDI-resistant polymeric misfolded protein forms that accumulate upon exposure to oxidative stress. Our study suggests that curcumin and masoprocol can serve as lead-candidate prophylactics for reactive oxygen species induced chaperone damage, protein misfolding and neurodegenerative disease; importantly, they can play a vital role in sustaining traffic along the ER’s secretory pathway by preserving functional integrity of PDI. 相似文献