FK506-binding protein of the hyperthermophilic archaeum, Thermococcus sp. KS-1, a cold-shock-inducible peptidyl-prolyl cis–trans isomerase with activities to trap and refold denatured proteins

2001 ◽  
Vol 357 (2) ◽  
pp. 465-471
Author(s):  
Akira IDENO ◽  
Takao YOSHIDA ◽  
Toshii IIDA ◽  
Masahiro FURUTANI ◽  
Tadashi MARUYAMA
Keyword(s):  
Citrate Synthase ◽  
Binding Protein ◽  
Binding Pocket ◽  
Molar Ratio ◽  
Hydrophobic Region ◽  
Native Form ◽  
Ppiase Activity ◽  
Molar Ratios ◽  
Multimeric Complex

The FK506 (tacrolimus)-binding protein (FKBP) type peptidyl-prolyl cis–trans isomerase (PPIase) in the hyperthermophilic archaeum Thermococcus sp. KS-1 was shown to be induced by temperature downshift to growth temperatures lower than the optimum. This PPIase (TcFKBP18) showed chaperone-like protein refolding activity in addition to PPIase activity in vitro. It refolded unfolded citrate synthase (CS) and increased the yield of the refolded protein. At a molar ratio of 15:1 ([TcFKBP18] to [CS]) in the refolding mixture, the recovered yield of folded CS was maximal at 62%, whereas that of spontaneous refolding was 11%. Increasing FKBP above a 15:1 ratio decreased the final yield, whereas the aggregation of unfolded CS was suppressed. A cross-linking analysis showed the formation of a complex between TcFKBP18 and unfolded CS (1:1 complex) at molar ratios of 3:1 to 15:1. However, molar ratios of 15:1 or 60:1 induced the binding of multiple FKBP molecules to an unfolded CS molecule (multimeric complex). Disrupting hydrophobic interaction by adding ethylene glycol at a molar ratio of 60:1 ([TcFKBP18] to [CS]) suppressed the formation of this multimeric complex, simultaneously enhancing CS refolding. FK506 also suppressed the formation of the multimeric complex while increasing the chaperone-like activity. These results suggest that the hydrophobic region of TcFKBP18, probably the FK506-binding pocket, was important for the interaction with unfolded proteins. No cross-linked product was detected between TcFKBP18 and native dimeric CS. TcFKBP18 probably traps the unfolded protein, then refolds and releases it in a native form. This FKBP might be important at growth temperatures lower than the optimum in Thermococcus sp. KS-1 cells.

scholarly journals Characterization of kinectin, a kinesin-binding protein: primary sequence and N-terminal topogenic signal analysis.

1995 ◽  
Vol 6 (2) ◽  
pp. 171-183 ◽  
Author(s):  
H Yu ◽  
C V Nicchitta ◽  
J Kumar ◽  
M Becker ◽  
I Toyoshima ◽  
...  
Keyword(s):  
Amino Acids ◽  
Transmembrane Domain ◽  
Binding Protein ◽  
Coiled Coil ◽  
Integral Membrane Protein ◽  
In Vitro Translation ◽  
Predicted Amino Acid Sequence ◽  
Hydrophobic Region ◽  
Reading Frame

Kinectin is a kinesin-binding protein (Toyoshima et al., 1992) that is required for kinesin-based motility (Kumar et al., 1995). A kinectin cDNA clone containing a 4.7-kilobase insert was isolated from an embryonic chick brain cDNA library by immunoscreening with a panel of monoclonal antibodies. The cDNA contained an open reading frame of 1364 amino acids encoding a protein of 156 kDa. A bacterially expressed product of the full length cDNA bound purified kinesin. Transient expression in CV-1 cells gave an endoplasmic reticulum distribution that depended upon the N-terminal domain. Analysis of the predicted amino acid sequence indicated a highly hydrophobic near N-terminal stretch of 28 amino acids and a large portion (326-1248) of predicted alpha helical coiled coils. The 30-kDa fragment containing the N-terminal hydrophobic region was produced by cell-free in vitro translation and found to assemble with canine pancreas rough microsomes. Cleavage of the N terminus was not observed confirming its role as a potential transmembrane domain. Thus, the kinectin cDNA encodes a cytoplasmic-oriented integral membrane protein that binds kinesin and is likely to be a coiled-coil dimer.

scholarly journals Size-Dependent Photodynamic Activity of Gold Nanoparticles Conjugate of Water Soluble Purpurin-18-N-Methyl-D-Glucamine

2013 ◽  
Vol 2013 ◽  
pp. 1-10 ◽  
Author(s):  
Byambajav Lkhagvadulam ◽  
Jung Hwa Kim ◽  
Il Yoon ◽  
Young Key Shim
Keyword(s):  
Gold Nanoparticles ◽  
A549 Cells ◽  
Water Soluble ◽  
Molar Ratio ◽  
Anticancer Efficacy ◽  
Molar Ratios ◽  
Size Dependent ◽  
Photodynamic Activity ◽  
Wavelength Absorption

Gold nanoparticles (GNPs) conjugates of water soluble ionic photosensitizer (PS), purpurin-18-N-methyl-D-glucamine (Pu-18-NMGA), were synthesized using various molar ratios between HAuCl4and Pu-18-NMGA without adding any particular reducing agents and surfactants. The PS-GNPs conjugates showed long wavelength absorption of range 702–762 nm, and their different shapes and diameters depend on the molar ratios used in the synthesis.In vitroanticancer efficacy of the PS-GNPs conjugates was investigated by MTT assay against A549 cells, resulting in higher photodynamic activity than that of the free Pu-18-NMGA. Among the PS-GNPs conjugates, the GNPs conjugate from the molar ratio of 1 : 2 (Au(III): Pu-18-NMGA) exhibits the highest photodynamic activity corresponding to bigger size (~60 nm) of the GNPs conjugate which could efficiently transport the PS into the cells than that of smaller size of the GNPs conjugate.

scholarly journals Improvement of Immunogenicity of Meningococcal Lipooligosaccharide by Coformulation with Lipidated Transferrin-Binding Protein B in Liposomes: Implications for Vaccine Development

2012 ◽  
Vol 19 (5) ◽  
pp. 711-722 ◽  
Author(s):  
Noëlle Mistretta ◽  
Bruno Guy ◽  
Yves Bérard ◽  
François Dalençon ◽  
Olivia Fratantonio ◽  
...  
Keyword(s):  
Vaccine Development ◽  
Binding Protein ◽  
Hek293 Cells ◽  
Molar Ratio ◽  
Adjuvant Effect ◽  
Content Type ◽  
Endotoxic Activity ◽  
Protein B

ABSTRACTAmong various meningococcal antigens, lipooligosaccharide (LOS) and recombinant lipidated transferrin-binding protein B (rlip-TbpB) are considered to be putative vaccine candidates against group BNeisseria meningitidis. In the present work, we report the development of a new liposome-based vaccine formulation containing both rlip-TbpB and L8 LOS. The endotoxic activity of the liposomal LOS was evaluatedin vitrousing theLimulusAmebocyte Lysate assay and compared to the endotoxic activity of free LOS. Above a 250:1 lipid/LOS molar ratio, liposomes were shown to effectively detoxify the LOS as the endotoxic activity of the LOS was reduced by more than 99%. Immunogenicity studies in rabbits showed that the presence of rlip-TbpB dramatically increased the immunogenicity of the LOS. While the formulation raised a strong anti-TbpB response, it elicited a higher anti-LOS IgG level than the liposomal LOS alone. Sera from rabbits immunized with rlip-TbpB/liposomal LOS displayed increased ability to recognize LOS on live bacteria expressing the L8 immunotype and increased anti-LOS-specific bactericidal activity compared to sera from rabbits immunized with liposomal LOS alone. Measurement of interleukin-8 (IL-8) produced by HEK293 cells transfected with Toll-like receptor (TLR) after stimulation with rlip-TbpB showed that the protein is a TLR2 agonist, which is in accordance with the structure of its lipid. Furthermore, anin vivostudy demonstrated that the lipid moiety is not only required for its adjuvant effect but also has to be linked to the protein. Overall, the rlip-TbpB/LOS liposomal formulation was demonstrated to induce an effective anti-LOS response due to the adjuvant effect of rlip-TbpB on LOS.

INTRAARTERIAL THROMBOLYSIS BY STREPTOKINASE-GLU-PLASMINOGEN

1987 ◽  
Author(s):  
E J Pilger ◽  
J Lammer ◽  
H Bertuch ◽  
H Steiner
Keyword(s):  
Artery Occlusion ◽  
Molar Ratio ◽  
In Vitro Test ◽  
Group Iii ◽  
Fibrinolytic Agent ◽  
Molar Ratios ◽  
Intraarterial Thrombolysis ◽  
Group I

In order to predict usefulness of streptokinase (SK), urokinase (UK) and streptokinase-glutamin-plasminogen (SK-Glu-Plg) for intraarterial fibrinolysis, an in vitro test was designed. Fibrin plates with and without plasminogen were incubated with SK, UK and SK-Glu-Plg (in molar ratios of 1:1, 2:1 and 1:2). On the fibrin plates containing plasminogen the highest fibrinolytic activity was observed with UK; on the fibrin plates without plasminogen, SK-Glu-Plg in a molar ratio of 1:2 was superior. We concluded, that plasmin would be synthesized by SK and Glu-Plg. In order to examine the in vivo efficacy of these different fibrinolytic agents, 120 patients suffering from peripheral artery occlusion were randomized into three treatment groups for local thrombolysis. The technique of Hess and coworkers was used for local thrombolytic therapy. In group I local fibrinolysis was performed with SK (2500 IU/5 min), in group II UK (4000 IU/5 min) was used and in group III the lytic agent consisted of SK-Glu-Plg (2500 IU/5 min). The primary recanalization rate was equal in all groups: 86%, 89% and 83% in group I, II and III respectively. However the duration required for the procedure and thus the total amount of fibrinolytic agent used was significantly different (p < 0,001) between the three groups: 2,3 ± 1,4; 2,1 ± 1,2 and 1,1 ±0,8 hours for groups I,II and III, respectively (mean ± SEM) . We conclude that SK-Glu-Plg or plasmin itself has the highest efficacy as a fibrinolytic agent for intraarterial thrombolysis. Since the intra-thrombotic concentration of Pig is unknown in an individual patient an empirically chosen dose of SK of UK may either be to high or to low for optimal thrombolysis.

scholarly journals FK506 Binding Protein from the Hyperthermophilic Archaeon Pyrococcus horikoshii Suppresses the Aggregation of Proteins in Escherichia coli

2002 ◽  
Vol 68 (2) ◽  
pp. 464-469 ◽  
Author(s):  
Akira Ideno ◽  
Masahiro Furutani ◽  
Yoshitaka Iba ◽  
Yoshikazu Kurosawa ◽  
Tadashi Maruyama
Keyword(s):  
Escherichia Coli ◽  
Protein Folding ◽  
Binding Protein ◽  
Fab Fragment ◽  
Ppiase Activity ◽  
Pyrococcus Horikoshii ◽  
E Coli ◽  
Fk506 Binding Protein ◽  
Soluble Recombinant Protein

ABSTRACT The 29-kDa FK506 binding protein (FKBP) gene is the only peptidyl-prolyl cis-trans isomerase (PPIase) gene in the genome of Pyrococcus horikoshii. We characterized the function of this FKBP (PhFKBP29) and used it to increase the production yield of soluble recombinant protein in Escherichia coli. The PPIase activity (k cat/Km ) of PhFKBP29 was found to be much lower than that of other archaeal 16- to 18-kDa FKBPs by a chymotrypsin-coupled assay of the oligo-peptidyl substrate at 15�C. Besides this low PPIase activity, PhFKBP29 showed chaperone-like protein folding activity which enhanced the refolding yield of chemically unfolded rhodanese in vitro. In addition, it suppressed thermal protein aggregation in a temperature range of 45 to 100�C. When the PhFKBP29 gene was coexpressed with the recombinant Fab fragment gene of the anti-hen egg lysozyme antibody in the cytoplasm of E. coli, whose expressed product tended to form an inactive aggregate in E. coli, it improved the yield of the soluble Fab fragments with antibody specificity. PhFKBP29 exerted protein folding and aggregation suppression in E. coli cells.

scholarly journals Hydrogel Films Based on Chitosan and Oxidized Carboxymethylcellulose Optimized for the Controlled Release of Curcumin with Applications in Treating Dermatological Conditions

Molecules ◽  
2021 ◽  
Vol 26 (8) ◽  
pp. 2185
Author(s):  
Mohamed Dellali ◽  
Camelia Elena Iurciuc (Tincu) ◽  
Corina Lenuța Savin ◽  
Nawel Spahis ◽  
M’hamed Djennad ◽  
...  
Keyword(s):  
Carboxymethyl Cellulose ◽  
Molar Ratio ◽  
Cross Linking ◽  
Oxidation Degree ◽  
Molar Ratios ◽  
Receptor Compartment ◽  
Index Value ◽  
Aldehyde Groups ◽  
Hydrogel Films

Cross-linked chitosan (CS) films with aldehyde groups obtained by oxidation of carboxymethyl cellulose (CMC) with NaIO4 were prepared using different molar ratios between the CHO groups from oxidized carboxymethyl cellulose (CMCOx) and NH2 groups from CS (from 0.25:1 to 2:1). Fourier-transform infrared (FTIR) and nuclear magnetic resonance (NMR) spectroscopy demonstrated the aldehyde groups’ presence in the CMCOx. The maximum oxidation degree was 22.9%. In the hydrogel, the amino groups’ conversion index value increased when the -CHO/-NH2 molar ratio, cross-linking temperature, and time increased, while the swelling degree values decreased. The hydrogel films were characterized by scanning electron microscopy (SEM) and FTIR analysis. The curcumin encapsulation efficiency decreases from 56.74% to 16.88% when the cross-linking degree increases. The immobilized curcumin release efficiency (REf%) and skin membrane permeability were evaluated in vitro in two different pH solutions using a Franz diffusion cell, and it was found to decrease when the molar ratio -CH=O/NH2 increases. The curcumin REf% in the receptor compartment was higher at pH = 7.4 (18%- for the sample with a molar ratio of 0.25:1) than at pH = 5.5 (16.5%). The curcumin absorption in the skin membrane at pH = 5.5 (47%) was more intense than at pH = 7.4 (8.6%). The curcumin-loaded films’ antioxidant activity was improved due to the CS presence.

scholarly journals Short-chain fatty acids produced in vitro from fibre residues obtained from mixed diets containing different breads and in human faeces during the ingestion of the diets

2000 ◽  
Vol 84 (1) ◽  
pp. 31-37 ◽  
Author(s):  
Elisabeth Wisker ◽  
Martina Daniel ◽  
Gerhard Rave ◽  
Walter Feldheim
Keyword(s):  
Fatty Acids ◽  
Short Chain Fatty Acids ◽  
Short Chain ◽  
Molar Ratio ◽  
Molar Ratios ◽  
Fibre Diet ◽  
The Difference ◽  
Wholemeal Bread ◽  
Chain Fatty Acids

It was studied whether the type of bread (i.e. a low-fibre wheat–rye mixed bread and coarse or fine wholemeal rye bread) either as part of a diet or alone, had an influence on the short-chain fatty acids (SCFA) produced during in vitro fermentation. Fermentation substrates were dietary fibre residues obtained from diets and breads. In addition, it was investigated whether the faecal SCFA pattern in the inoculum donors, who ingested the experimental diets, could be predicted by in vitro fermentation. Yields of SCFA in vitro were 0·51–0·62 g/g fermented polysaccharide. In vitro, the molar ratios of butyrate were higher for the two high-fibre diets containing coarse or fine wholemeal bread than for the low fibre diet containing wheat–rye mixed bread; the difference was significant for the coarse (P < 0·01), but not for the fine bread diet (P = 0·0678). The coarse wholemeal bread alone produced a higher molar ratio of butyrate than the fine wholemeal bread (P < 0·05) and the wheat–rye mixed bread (P < 0·01). Ingestion by the inoculum donors of the diets containing wholemeal bread led to higher faecal butyrate ratios (molar ratios: coarse bread diet 19·6, fine bread diet 17·7) compared with the wheat–rye mixed bread-containing diet (14·9), but the differences between the diets were not significant. For the diets investigated, there were no significant differences between faecal and in vitro SCFA patterns.

Effect of FKBP65, a putative elastin chaperone, on the coacervation of tropoelastin in vitro

2010 ◽  
Vol 88 (6) ◽  
pp. 917-925 ◽  
Author(s):  
Kevin L.Y. Cheung ◽  
Matthew Bates ◽  
Vettai S. Ananthanarayanan
Keyword(s):  
Self Assembly ◽  
Secretory Pathway ◽  
Molar Ratio ◽  
Prolyl Isomerase ◽  
Ppiase Activity ◽  
Peptidyl Prolyl Isomerase ◽  
Maturation Stages ◽  
Turbidimetric Assay

FKBP65 is a protein of the endoplasmic reticulum that is relatively abundant in elastin-producing cells and is associated with tropoelastin in the secretory pathway. To test an earlier suggestion by Davis and co-workers that FKBP65 could act as an intracellular chaperone for elastin, we obtained recombinant FKBP65 (rFKBP65) by expressing it in E. coli and examined its effect on the coacervation characteristics of chicken aorta tropoelastin (TE) using an in vitro turbidimetric assay. Our results reveal that rFKBP65 markedly promotes the initiation of coacervation of TE without significantly affecting the temperature of onset of coacervation. This effect shows saturation at a 1:2 molar ratio of TE to rFKBP65. By contrast, FKBP12, a peptidyl prolyl isomerase, has a negligible effect on TE coacervation. Moreover, the effect of rFKBP65 on TE coacervation is unaffected by the addition of rapamycin, an inhibitor of peptidyl prolyl isomerase (PPIase) activity. These observations rule out the involvement of the PPIase activity of rFKBP65 in modulating the coacervation of TE. Additional experiments using a polypeptide model of TE showed that rFKBP65, while promoting coacervation, may retard the maturation of this model polypeptide into larger aggregates. Based on these results, we suggest that FKBP65 may act as an elastin chaperone in vivo by controlling both the coacervation and the maturation stages of its self-assembly into fibrils.

scholarly journals Single-Domain Peptidyl-Prolylcis/transIsomerase FkpA from Corynebacterium glutamicum Improves the Biomass Yield at Increased Growth Temperatures

2015 ◽  
Vol 81 (22) ◽  
pp. 7839-7850 ◽  
Author(s):  
Nicolai Kallscheuer ◽  
Michael Bott ◽  
Jan van Ooyen ◽  
Tino Polen
Keyword(s):  
Corynebacterium Glutamicum ◽  
Biomass Yield ◽  
Citrate Synthase ◽  
Mrna Level ◽  
Single Domain ◽  
Artificial Substrates ◽  
Ppiase Activity ◽  
Fk 506 ◽  
Content Type

ABSTRACTPeptidyl-prolylcis/transisomerases (PPIases) catalyze the rate-limiting protein folding step at peptidyl bonds preceding proline residues and were found to be involved in several biological processes, including gene expression, signal transduction, and protein secretion. Representative enzymes were found in almost all sequenced genomes, includingCorynebacterium glutamicum, a facultative anaerobic Gram-positive and industrial workhorse for the production of amino acids. InC. glutamicum, a predicted single-domain FK-506 (tacrolimus) binding protein (FKBP)-type PPIase (FkpA) is encoded directly downstream ofgltA, which encodes citrate synthase (CS). This gene cluster is also present in otherActinobacteria. Here we carried outin vitroandin vivoexperiments to study the function and influence of predicted FkpA inC. glutamicum.In vitro, FkpA indeed shows typical PPIase activity with artificial substrates and is inhibited by FK-506. Furthermore, FkpA delays the aggregation of CS, which is also inhibited by FK-506. Surprisingly, FkpA has a positive effect on the activity and temperature range of CSin vitro. Deletion offkpAcauses a 50% reduced biomass yield compared to that of the wild type when grown at 37°C, whereas there is only a 10% reduced biomass yield at the optimal growth temperature of 30°C accompanied by accumulation of 7 mMl-glutamate and 22 mM 2-oxoglutarate. Thus, FkpA may be exploited for improved product formation in biotechnical processes. Comparative transcriptome analysis revealed 69 genes which exhibit ≥2-fold mRNA level changes inC. glutamicumΔfkpA, giving insight into the transcriptional response upon mild heat stress when FkpA is absent.

scholarly journals SELECTION OF PHOSPHOLIPID AND METHOD OF FORMULATION FOR OPTIMUM ENTRAPMENT AND RELEASE OF LAMIVUDINE FROM LIPOSOME

2018 ◽  
Vol 8 (5-s) ◽  
pp. 175-183 ◽  
Author(s):  
Mangesh D Godbole ◽  
Vijay B Mathur
Keyword(s):  
Thin Film ◽  
Encapsulation Efficiency ◽  
In Vitro Release ◽  
Molar Ratio ◽  
Vesicle Size ◽  
Injection Method ◽  
Molar Ratios ◽  
Vitro Release ◽  
Selection Of

The present investigation was aimed to compare various phospholipids and different methods that would be appropriate to produce liposomes having vesicle size in the range of 200-300 nm, PDI less than 0.500, maximum entrapment and delayed release of lamivudine. The phospholipids employed were Phospholipon® 90G, Phospholipon® 90H, 1,2-Dimyristoyl-sn-glysero-3-phosphocholine (DMPC) and 1,2-Dipalmitoyl-sn-glysero-3-phosphocholine (DPPC). They were used in various molar ratio with cholesterol and blank liposomes were prepared initially by thin film hydration and ether injection method. The thin film hydration method was only found to be appropriate to produce liposome of desired size and PDI. Hence, the molar ratios of employed phospholipids:cholesterol that produced liposomes as per the expected parameters was then used to load lamivudine. The method of preparation, phospholipid: cholesterol molar ratio, hydrations above transition temperature and hydration time were showed direct influence on vesicle size, PDI, percentage encapsulation and in-vitro release. Phospholipon® 90H: cholesterol: lamivudine in the molar ratio 1:2:1 produced liposomes having desired vesicle size and PDI with maximum drug entrapment and sustained release. The encapsulation efficiency of drug in liposomes was in the order of Phospholipon® 90H> Phospholipon® 90G > DPPC > DMPC. Keywords: Liposomes, Lamivudine, Phospholipid, Thin film hydration method, Ether injection method, encapsulation efficiency

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