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Human Serum Albumin (human + serum_albumin)
Selected AbstractsMolecular Interaction between a Gadolinium,Polyoxometalate and Human Serum AlbuminEUROPEAN JOURNAL OF INORGANIC CHEMISTRY, Issue 34 2009Li Zheng Abstract Polyoxometalates (POMs) show promising antibacterial, antiviral (particularly anti-HIV), antitumor, and anticancer activities, but the mechanism of these potential therapeutic effects remains to be elucidated at the molecular level. The interaction between the Gd-containing tungstosilicate [Gd(,2 -SiW11O39)2]13, and human serum albumin (HSA) was studied by several techniques. Fluorescence spectroscopy showed an energy transfer between the single tryptophan residue of HSA and the POM. Circular dichroism led to the conclusion that the POM significantly altered the secondary structure of HSA. Isothermal titration calorimetry revealed an enthalpy-driven binding reaction between HSA and the POM, resulting in the formation of a 1:1 complex.(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2009) [source] Binding of Warfarin Influences the Acid-Base Equilibrium of H242 in Sudlow Site I of Human Serum AlbuminPHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 5 2006Jennifer L. Perry ABSTRACT Sudlow Site I of human serum albumin (HSA) is located in subdomain IIA of the protein and serves as a binding cavity for a variety of ligands. In this study, the binding of warfarin (W) is examined using computational techniques and isothermal titration calorimetry (ITC). The structure of the docked warfarin anion (W,) to Site I is similar to that revealed by X-ray crystallography, with a calculated binding constant of 5.8 × 105M,1. ITC experiments (pH 7.13 and I = 0.1) carried out in three different buffers (MOPs, phosphate and Tris) reveal binding of W, is accompanied by uptake of 0.30 ± 0.02 protons from the solvent. This measurement suggests that the binding of W, is stabilized by an ion-pair interaction between protonated H242 and the phenoxide group of W,. [source] A chloroplast transgenic approach to hyper-express and purify Human Serum Albumin, a protein highly susceptible to proteolytic degradationPLANT BIOTECHNOLOGY JOURNAL, Issue 2 2003Alicia Fernández-San Millán Summary Human Serum Albumin (HSA) accounts for 60% of the total protein in blood serum and it is the most widely used intravenous protein in a number of human therapies. HSA, however, is currently extracted only from blood because of a lack of commercially feasible recombinant expression systems. HSA is highly susceptible to proteolytic degradation in recombinant systems and is expensive to purify. Expression of HSA in transgenic chloroplasts using Shine-Dalgarno sequence (SD), which usually facilitates hyper-expression of transgenes, resulted only in 0.02% HSA in total protein (tp). Modification of HSA regulatory sequences using chloroplast untranslated regions (UTRs) resulted in hyper-expression of HSA (up to 11.1% tp), compensating for excessive proteolytic degradation. This is the highest expression of a pharmaceutical protein in transgenic plants and 500-fold greater than previous reports on HSA expression in transgenic leaves. Electron micrographs of immunogold labelled transgenic chloroplasts revealed HSA inclusion bodies, which provided a simple method for purification from other cellular proteins. HSA inclusion bodies could be readily solubilized to obtain a monomeric form using appropriate reagents. The regulatory elements used in this study should serve as a model system for enhancing expression of foreign proteins that are highly susceptible to proteolytic degradation and provide advantages in purification, when inclusion bodies are formed. [source] The Structure of the Sugar Residue in Glycated Human Serum Albumin and Its Molecular Recognition by Phenylboronate.CHEMINFORM, Issue 32 2003Jan Rohovec No abstract is available for this article. [source] A High Performance Theory for Thermodynamic Study on the Binding of Human Serum Albumin with Erbium ChlorideCHINESE JOURNAL OF CHEMISTRY, Issue 2 2009G. Rezaei BEHBEHANI Abstract A thermodynamic study of the interaction between erbium(III) chloride (Er3+) and human serum albumin (HSA) was studied at pH=7.0, 27 and 37 °C in phosphate buffer by isothermal titration calorimetry (ITC). The present study reports the thermodynamic parameters that govern HSA-Er3+ interactions. The extended solvation theory was used to reproduce the enthalpies of HSA-Er3+ interactions over the whole range of Er3+ concentrations. The binding parameters recovered from the new model were attributed to the structural change of HSA and its biological activity. The results obtained indicate that there is a set of two identical binding sites for Er3+ ions with negative cooperativity. The enhancement of complex formation by Er3+ and concomitant increase in ?S suggest that the metal ion plays a role in increasing the number of hydrophobic contacts. The binding parameters discovered from the extended solvation model indicate that the stability of HSA molecule is increased as a result of its interaction with Er3+ ions. [source] Study on Interaction of Ginsenosides with Bovine or Human Serum Albumin Using Wavelength Modulation Surface Plasmon Resonance BiosensorCHINESE JOURNAL OF CHEMISTRY, Issue 5 2006Xia Liu Abstract To use a newly developed wavelength modulation surface plasmon resonance (SPR) biosensor, an experimental protocol was developed to investigate the interaction of ginsenosides with serum albumin. With a known concentration of the ginsenosides, bound percentages of the ginsenosides with human serum albumin (HSA) or bovine serum albumin (BSA) were obtained. SPR technique could require no labeling and this method provided the detailed information on association and disassociation of molecules in real time. The results indicate that the sensitivity of wavelength modulation SPR biosensor is sufficient for detection and characterization of binding events involving low-molecular weight compounds and their immobilized protein targets. [source] Allosteric modulation of anti-HIV drug and ferric heme binding to human serum albuminFEBS JOURNAL, Issue 24 2005Alessio Bocedi Human serum albumin (HSA), the most prominent protein in plasma, is best known for its exceptional capacity to bind ligands (e.g. heme and drugs). Here, binding of the anti-HIV drugs abacavir, atazanavir, didanosine, efavirenz, emtricitabine, lamivudine, nelfinavir, nevirapine, ritonavir, saquinavir, stavudine, and zidovudine to HSA and ferric heme,HSA is reported. Ferric heme binding to HSA in the absence and presence of anti-HIV drugs was also investigated. The association equilibrium constant and second-order rate constant for the binding of anti-HIV drugs to Sudlow's site I of ferric heme,HSA are lower by one order of magnitude than those for the binding of anti-HIV drugs to HSA. Accordingly, the association equilibrium constant and the second-order rate constant for heme binding to HSA are decreased by one order of magnitude in the presence of anti-HIV drugs. In contrast, the first-order rate constant for ligand dissociation from HSA is insensitive to anti-HIV drugs and ferric heme. These findings represent clear-cut evidence for the allosteric inhibition of anti-HIV drug binding to HSA by the heme. In turn, anti-HIV drugs allosterically impair heme binding to HSA. Therefore, Sudlow's site I and the heme cleft must be functionally linked. [source] The extraordinary ligand binding properties of human serum albuminIUBMB LIFE, Issue 12 2005Mauro Fasano Abstract Human serum albumin (HSA), the most prominent protein in plasma, binds different classes of ligands at multiple sites. HSA provides a depot for many compounds, affects pharmacokinetics of many drugs, holds some ligands in a strained orientation providing their metabolic modification, renders potential toxins harmless transporting them to disposal sites, accounts for most of the antioxidant capacity of human serum, and acts as a NO-carrier. The globular domain structural organization of monomeric HSA is at the root of its allosteric properties which are reminiscent of those of multimeric proteins. Here, structural, functional, biotechnological, and biomedical aspects of ligand binding to HSA are summarized. [source] Separation with zwitterionic hydrophilic interaction liquid chromatography improves protein identification by matrix-assisted laser desorption/ionization-based proteomic analysisBIOMEDICAL CHROMATOGRAPHY, Issue 6 2009Atsushi Intoh Abstract Comprehensive proteomic analyses necessitate efficient separation of peptide mixtures for the subsequent identification of proteins by mass spectrometry (MS). However, digestion of proteins extracted from cells and tissues often yields complex peptide mixtures that confound direct comprehensive MS analysis. This study investigated a zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) technique for the peptide separation step, which was verified by subsequent MS analysis. Human serum albumin (HSA) was the model protein used for this analysis. HSA was digested with trypsin and resolved by ZIC-HILIC or conventional strong cation exchange (SCX) prior to MS analysis for peptide identification. Separation with ZIC-HILIC significantly improved the identification of HSA peptides over SCX chromatography. Detailed analyses of the identified peptides revealed that the ZIC-HILIC has better peptide fractionation ability. We further demonstrated that ZIC-HILIC is useful for quantitatively surveying cell surface markers specifically expressed in undifferentiated embryonic stem cells. These results suggested the value of ZIC-HILIC as a novel and efficient separation method for comprehensive and quantitative proteomic analyses. Copyright © 2009 John Wiley & Sons, Ltd. [source] Affinity Ligand Selection from a Library of Small Molecules: Assay Development, Screening, and ApplicationBIOTECHNOLOGY PROGRESS, Issue 1 2005Lakshmi D. Saraswat A facile and cost-effective process for screening synthetic libraries for an affinity ligand is described. A high throughput 96-well plate filtration method was designed to screen both discrete compounds and mixtures of compounds attached to a solid support. Human serum albumin (HSA) was used as a target protein to demonstrate the proof of concept. Detection and quantitation by fluorescence was accomplished with the use of fluorescamine to conjugate the protein in the filtrate. It is found that mixtures demonstrating low average binding reflect an overall lower hit rate of the components, whereas deconvolution of mixtures with high protein binding consistently provides a high hit rate. This differs from many of the previous experiences screening solid-phase mixtures in which high false positive rates are noted to occur. A total of 100K compounds were tested: 25K as discrete samples and 75K as mixtures. An overall hit rate of 8% was observed. Secondary screening of compounds measured specificity, recovery, and dynamic binding capacity. The effectiveness of the method is illustrated using an affinity column made with a representative lead compound. A similar purity was achieved in a single-step purification of HSA from serum as compared to that obtained by two steps of ion-exchange chromatography. The process for primary screening of a large number of compounds is simple, inexpensive, and applicable to any soluble target protein of known or unknown function from crude mixtures and may have additional utility as a generic chemical affinity tool for the functional characterization of novel proteins emerging from proteomics work. [source] Ligand binding strategies of human serum albumin: How can the cargo be utilized?CHIRALITY, Issue 1 2010Ankita Varshney Abstract Human serum albumin (HSA), being the most abundant carrier protein in blood and a modern day clinical tool for drug delivery, attracts high attention among biologists. Hence, its unfolding/refolding strategies and exogenous/endogenous ligand binding preference are of immense use in therapeutics and clinical biochemistry. Among its fellow proteins albumin is known to carry almost every small molecule. Thus, it is a potential contender for being a molecular cargo/or nanovehicle for clinical, biophysical and industrial purposes. Nonetheless, its structure and function are largely regulated by various chemical and physical factors to accommodate HSA to its functional purpose. This multifunctional protein also possesses enzymatic properties which may be used to convert prodrugs to active therapeutics. This review aims to highlight current overview on the binding strategies of protein to various ligands that may be expected to lead to significant clinical applications. Chirality, 2010. © 2009 Wiley-Liss, Inc. [source] Stereoselective binding of human serum albuminCHIRALITY, Issue 3 2006Victor Tuan Giam Chuang Abstract Stereoselectivity in binding can have a significant effect on the drug disposition such as first-pass metabolism, metabolic clearance, renal clearance, and protein and tissue binding. Human serum albumin (HSA) is able to stereoselectively bind a great number of various endogenous and exogenous compounds. Various experimental data suggested that the two major drug-binding cavities, namely, site I and site II, do not seem to be the stereoselective binding sites of HSA. Stereoselective binding of HSA under disease conditions such as renal and hepatic diseases was found to be enhanced. In addition, site-to-site displacement of a site II-specific drug by another site II-specific drug was found to be stereoselective, too. Endogenous compounds such as long-chain fatty acids and uremic toxins are likely to cause combined direct and cascade effects that contribute to the preferential binding of a particular drug enantiomer. Taking together the findings of other studies, it is highly possible that the stereoselective binding site exists at the interface of the subdomains. © 2006 Wiley-Liss, Inc. Chirality [source] Molecular recognition of sugars by lanthanide (III) complexes of a conjugate of N, N -bis[2-[bis[2-(1, 1-dimethylethoxy)-2-oxoethyl]amino]ethyl]glycine and phenylboronic acidCONTRAST MEDIA & MOLECULAR IMAGING, Issue 4 2007Elisa Battistini Abstract A novel conjugate of phenylboronic acid and an Ln(DTPA) derivative, in which the central acetate pendant arm was replaced by the methylamide of L -lysine, was synthesized and characterized. The results of a fit of variable 17O NMR data and a 1H NMRD profile show that the water residence lifetime of the Gd(III) complex (150,ns) is shorter than that of the parent compound Gd(DTPA)2, (303,ns). Furthermore, the data suggest that several water molecules in the second coordination sphere of Gd(III) contribute to the relaxivity of the conjugate. The Ln(III) complexes of this conjugate are highly suitable for molecular recognition of sugars. The interaction with various sugars was investigated by 11B NMR spectroscopy. Thanks to the thiourea function that links the phenylboronic acid targeting vector with the DTPA derivative, the interactions are stronger than that of phenylboronic acid itself. In particular, the interaction with N -propylfructosamine, a model for the glucose residue in glycated human serum albumin (HSA), is very strong. Unfortunately, the complex also shows a rather strong interaction with hexose-free HSA (KA,=,705,±,300). Copyright © 2007 John Wiley & Sons, Ltd. [source] Electrochemically Induced Formation of Surface-Attached Temperature-Responsive Hydrogels.ELECTROANALYSIS, Issue 9 2010Amperometric Glucose Sensors with Tunable Sensor Characteristics Abstract Employing thermally responsive hydrogels, the design of an amperometric glucose sensor is proposed. The properties of the biosensor can be modulated upon changing the temperature. Homo- and copolymers of N -isopropylacrylamide (NIPAm) and oligo(ethylene glycol) methacrylate (OEGMA) were prepared by electrochemically induced polymerization thus yielding surface-attached hydrogels. The growth of the films as well as the change in the film thickness in dependence from the temperature were investigated by means of an electrochemical quartz crystal microbalance (EQCM). The layer thickness in the dry state ranged from 20 to 120,nm. The lower critical solution temperature (LCST) of the hydrogel increases with increasing content of the more hydrophilic OEGMA. Hence, the swelling in aqueous electrolyte is composition dependent and can be adjusted by selecting a specific NIPAm to OEGMA ratio. All homo- and copolymer films showed good biocompatibility and no fouling could be observed during exposing the surfaces to human serum albumin. For amperometric glucose detection, glucose oxidase was entrapped in the films during electrochemically-induced polymerization. Both the apparent Michaelis constant (K and the apparent maximum current (i as determined by amperometry could be adjusted both by the film composition as well as the operation temperature. [source] Characterization of interaction between doxycycline and human serum albumin by capillary electrophoresis-frontal analysisELECTROPHORESIS, Issue 11 2009Hanwen Sun Abstract The binding of doxycycline to HSA under simulated physiological conditions (pH 7.4, 67,mM phosphate, I=0.17, drug concentration 100,,M, HSA concentration up to 475,,M, 36.5°C) was studied by CE-frontal analysis. The number of primary binding sites, binding constant and physiological protein-binding percentage were 1.9, 1.51×103,M,1 and 59.80%, respectively. In addition, the thermodynamic parameters including enthalpy change (,H), entropy change (,S) and free energy change (,G) of the reaction were obtained in order to characterize the acting forces between doxycycline and HSA. Furthermore, to better understand the nature of doxycycline,HSA binding and to get information about potential interaction with other drugs, displacement experiments were performed. The results showed that doxycycline binds at site II of HSA. [source] Evaluation of enantioselective binding of antihistamines to human serum albumin by ACEELECTROPHORESIS, Issue 15 2007María Amparo Martínez-Gómez Abstract The drug binding to plasma and tissue proteins is a fundamental factor in determining the overall pharmacological activity of a drug. HSA, together with ,1 -acid glycoprotein, are the most important plasma proteins, which act as drug carriers, with implications on the pharmacokinetic of drugs. Among plasma proteins, HSA possesses the highest enantioselectivity. In this paper, a new methodology for the study of enantiodifferentiation of chiral drugs with HSA is developed and applied to evaluate the possible enantioselective binding of four antihistamines: brompheniramine, chlorpheniramine, hydroxyzine and orphenadrine to HSA. This study includes the determination of affinity constants of drug enantiomers to HSA and the evaluation of the binding sites of antihistamines on the HSA molecule. The developed methodology includes the ultrafiltration of samples containing HSA and racemic antihistaminic drugs and the analysis of the free or bound drug fraction using the affinity EKC-partial filling technique and HSA as chiral selector. The results shown in this paper represent the first evidence of the enantioselective binding of antihistamines to HSA, the major plasmatic protein. [source] Molecular Interaction between a Gadolinium,Polyoxometalate and Human Serum AlbuminEUROPEAN JOURNAL OF INORGANIC CHEMISTRY, Issue 34 2009Li Zheng Abstract Polyoxometalates (POMs) show promising antibacterial, antiviral (particularly anti-HIV), antitumor, and anticancer activities, but the mechanism of these potential therapeutic effects remains to be elucidated at the molecular level. The interaction between the Gd-containing tungstosilicate [Gd(,2 -SiW11O39)2]13, and human serum albumin (HSA) was studied by several techniques. Fluorescence spectroscopy showed an energy transfer between the single tryptophan residue of HSA and the POM. Circular dichroism led to the conclusion that the POM significantly altered the secondary structure of HSA. Isothermal titration calorimetry revealed an enthalpy-driven binding reaction between HSA and the POM, resulting in the formation of a 1:1 complex.(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2009) [source] An X-ray Spectromicroscopy Study of Albumin Adsorption to Crosslinked Polyethylene Oxide Films,ADVANCED ENGINEERING MATERIALS, Issue 5 2010Bonnie O. Leung Abstract Synchrotron-based X-ray photoemission electron microscopy (X-PEEM) is used to characterize the near surface composition of polyethylene oxide (PEO) combined with 1.5, 5, and 10,wt.-% pentaerythritol triacrylate (PETA) crosslinker. It is found that as the concentration of PETA increases, it becomes the dominant component in the top 10,nm of the film surface. The same surfaces are also exposed to human serum albumin (HSA) and the distributions of the protein relative to PEO and PETA measured with X-PEEM. A positive correlation is found between levels of PETA and HSA at the surface. Above PETA concentrations of 5,wt.-%, HSA adsorption is significant, which suggests high levels of PETA (often used to immobilize PEO by crosslinking) can significantly reduce the non-fouling properties of PEO. [source] Peptides of human gingival crevicular fluid determined by HPLC-ESI-MSEUROPEAN JOURNAL OF ORAL SCIENCES, Issue 6 2005Elisabetta Pisano The acidic-soluble protein content of human gingival crevicular fluid was analyzed by reverse-phase high-performance liquid chromatography (RP-HPLC), and the eluent deriving from the chromatography separation was directly introduced into an ion-trap mass spectrometer through electrospray ionization (ESI-IT MS). By this technique the molecular weight of peptides/proteins was determined with a precision of ,,1/10,000 amu. On the basis of the chromatographic behavior and the knowledge of the molecular mass value, some peptides and proteins soluble in acidic solution were unambiguously recognized. Besides high quantities of human serum albumin, , -defensins 1,4 and minor amounts of cystatin A, statherin, basic PB salivary peptide and other unidentified components were detected. The presence of , -defensins in gingival crevicular fluid is in agreement with their relevant contribution to protein composition deriving from granulocyte secretions. Other peptides and proteins abundant in human saliva, such as proline-rich proteins (PRPs) and histatins, were not detected in gingival crevicular fluid. Further investigations will be necessary to establish the origin of statherin and PB salivary peptide in gingival crevicular fluid. [source] Systematic Studies on Photoluminescence of Oligo(arylene-ethynylene)s: Tunability of Excited States and Derivatization as Luminescent Labeling Probes for ProteinsEUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 14 2006Yong-Gang Zhi Abstract Functionalized oligo(phenylene-ethynylene)s (OPEs) with different conjugation lengths, p -X(C6H4C,C)nSiMe3 (n = 1,4; X = NH2, NMe2, H) were synthesized by Sonogashira coupling of (phenylene-ethynylene)s and 1-iodo-4-(trimethylsilylethynyl)benzene, followed by desilylation of the p -substituted (trimethylsilylethynyl)benzenes with potassium hydroxide. The photoluminescent properties for the OPE series with different chain lengths and their solvatochromic responses were examined. The absorption maxima were red-shifted with increasing numbers of ,(C6H4C,C), units (n), and a linear plot of the absorption energy maxima vs. 1/n was obtained for each series. The emission spectra in dichloromethane showed a broad and structureless band, the energies of which (in wavenumbers) also fit linearly with 1/n. Both the absorption and emission wavelength maxima of the NH2 - and NMe2 -substituted OPEs exhibited significant solvent dependence, whereas the parent OPEs (X = H) showed only minor shifts of the ,max values in different solvents. Substituent effects upon the photoluminescent characteristics of the OPEs and the tunability of the excited states were examined with the p -X(C6H4C,C)nSiMe3 (n = 2, 3; X = NH2, NMe2, H, SMe, OMe, OH, and F) series. The H- and F-substituted counterparts exhibited high-energy vibronically structured emissions attributed to the 3(,,*) excited states of the (arylene-ethynylene) backbone. For compounds bearing NH2 and NMe2 groups, a broad red-shifted emission with a remarkable Stokes shift from the respective absorption maximum was observed, which can be assigned to an n , ,* transition. The n , ,* assignment was supported by MO calculations on the model compounds p -X(C6H4C,C)2SiH3 (X = NH2, H). Functionalization of the oligo(arylene-ethynylene)s with the N -hydroxysuccinimidyl (NHS) moiety enabled covalent attachment of the fluorophore to HSA protein molecules. A series of fluorescent labels, namely p -X(C6H4C,C)nC6H4NHS, (n = 1, X = NH2, NMe2, SMe, OMe, OH, F; n = 2, X = NH2, NMe2) and p -Me2NC6H4C,C(C4H2S)C,CC6H4NHS were synthesized, and their conjugates with HSA (human serum albumin) were characterized by MALDI-TOF mass spectrometry, UV/Vis absorption spectroscopy, and gel electrophoresis. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2006) [source] Allosteric modulation of anti-HIV drug and ferric heme binding to human serum albuminFEBS JOURNAL, Issue 24 2005Alessio Bocedi Human serum albumin (HSA), the most prominent protein in plasma, is best known for its exceptional capacity to bind ligands (e.g. heme and drugs). Here, binding of the anti-HIV drugs abacavir, atazanavir, didanosine, efavirenz, emtricitabine, lamivudine, nelfinavir, nevirapine, ritonavir, saquinavir, stavudine, and zidovudine to HSA and ferric heme,HSA is reported. Ferric heme binding to HSA in the absence and presence of anti-HIV drugs was also investigated. The association equilibrium constant and second-order rate constant for the binding of anti-HIV drugs to Sudlow's site I of ferric heme,HSA are lower by one order of magnitude than those for the binding of anti-HIV drugs to HSA. Accordingly, the association equilibrium constant and the second-order rate constant for heme binding to HSA are decreased by one order of magnitude in the presence of anti-HIV drugs. In contrast, the first-order rate constant for ligand dissociation from HSA is insensitive to anti-HIV drugs and ferric heme. These findings represent clear-cut evidence for the allosteric inhibition of anti-HIV drug binding to HSA by the heme. In turn, anti-HIV drugs allosterically impair heme binding to HSA. Therefore, Sudlow's site I and the heme cleft must be functionally linked. [source] Increase of calnexin gene dosage boosts the secretion of heterologous proteins by Hansenula polymorphaFEMS YEAST RESEARCH, Issue 7 2007Jens Klabunde Abstract The type I membrane protein calnexin is a conserved key component of the quality control mechanism in the endoplasmic reticulum. It functions as a molecular chaperone that monitors the folding state of nascent polypeptides entering the endoplasmic reticulum. Calnexin also behaves as a lectin, as its chaperoning activity involves binding of oligosaccharide moieties present on newly imported glycoproteins. We isolated the calnexin gene (HpCNE1) from the methylotrophic yeast Hansenula polymorpha, and used HpCNE1 expression plasmids for supertransformation of H. polymorpha strains secreting target proteins of biotechnological interest. The elevated dosage of HpCNE1 enhanced secretion of the four proteins tested: three glycoproteins and one unglycosylated product. Secretion of bacterial alginate epimerase AlgE1 was increased threefold on average, and secretion of both human interferon-, and fungal consensus phytase twofold. With phytase and AlgE1 this improvement was all the more remarkable, as the secretion level was already high in the original strains (g L,1 range). The same approach improved secretion of human serum albumin, which lacks N-linked glycans, about twofold. Glycosylation of the pro-MF,1 leader may account for the effect of calnexin in this case. Our results argue that cooverexpression of calnexin can serve as a generally applicable tool for enhancing the secretion of all types of heterologous protein by H. polymorpha. [source] REVIEW ARTICLE: An improved manufacturing process for Xyntha/ReFacto AFHAEMOPHILIA, Issue 5 2010B. KELLEY Summary., ReFacto® Antihemophilic Factor is a second-generation antihaemophilia A product manufactured using a process that includes therapeutic grade human serum albumin (HSA) in the cell culture medium, but is formulated without HSA as a stabilizer. Even though this second-generation antihaemophilia product has a good safety profile, a programme was implemented to eliminate all animal- and human-derived raw materials from the production process, thus producing a third-generation product. To that end, HSA has been removed from the master and working cell banks and from the culture medium. The hybridoma-derived monoclonal antibody formerly used in the purification process has been replaced by a chemically synthesized affinity peptide, and a virus-retaining filtration step has been added to enhance the clearance of large viruses, such as retroviruses. The purification process has been validated for the removal of a panel of model viruses and provides significant clearance of all viruses tested. Host cell- and process-derived impurity removal validations also were conducted, including host cell DNA and protein, in addition to the affinity peptide. Compared with the product manufactured according to the original process, these changes had no detectable effect on the structural integrity, stability or clinical efficacy of this antihaemophilia A product. The product produced by the improved manufacturing process is named XynthaÔ/ReFacto AF. [source] Safety and efficacy of a new recombinant FVIII formulated with sucrose (rFVIII,FS) in patients with haemophilia A: a long-term, multicentre clinical study in JapanHAEMOPHILIA, Issue 3 2001A. Yoshioka The recombinant full-length FVIII product Kogenate® has been reformulated using sucrose (rFVIII,FS) instead of human serum albumin as a stabiliser in purification and formulation. The in vivo recovery, haemostatic efficacy, and safety of rFVIII,FS were investigated in 20 previously treated patients with severe or moderate haemophilia A for , 24 weeks. In vivo recoveries of 73.5 ± 16.3%, 78.4 ± 16.1%, and 82.8 ± 23.9% after the initial infusion of 50 IU kg,1 rFVIII,FS and at weeks 12 and 24, respectively, showed no significant changes over time. A total of 1115 infusions (mean dose 24.1 ± 8.4 IU kg,1) were included in the analysis of haemostatic efficacy. One (80.5%) or two (8.2%) infusions achieved adequate haemostasis in 88.7% of all bleeding episodes, and haemostatic efficacy was judged ,excellent' or ,good' in 749 of 764 episodes (98.0%). The haemostatic efficacy was judged as ,excellent' or ,good' in 924 of 1115 (82.9%) infusions. Twenty-one adverse events were observed in 12 patients in the total 1541 infusions included in the safety analysis. Causality with respect to rFVIII,FS could not be ruled out in three events in one HIV-negative patient: elevated CD4(%), decreased CD8(%), and elevated CD4/CD8 ratio. No FVIII inhibitor development was observed in any patient. ELISA assay testing for antibodies to rFVIII, baby hamster kidney cell (BHK) protein, and murine IgG were all negative. These results show that rFVIII,FS is a safe and effective for long-term treatment of patients with haemophilia A. [source] Conjugation of methotrexate to immunoglobulins kills macrophages by Fc receptor mediated uptake?INTERNATIONAL JOURNAL OF LABORATORY HEMATOLOGY, Issue 3 2008X. WANG Summary The aim of this study was to conjugate methotrexate (MTX) with intravenous immunoglobulin (IVIG) and investigate whether the conjugate produce selective cytotoxicity on macrophages to provide a new strategy for the management of idiopathic thrombocytopenic purpura. MTX was bound to IVIG via human serum albumin as an intermediary. The binding activity of the Fc fragment of the conjugate was assayed by flow cytometry. The selective cytotoxicity of the conjugate was determined by trypan blue exclusion. After conjugating, the binding activity of the conjugate to Fc receptors did not diminish when compared with IVIG. In vitro, the conjugate showed significantly higher cytotoxicity to macrophages than Hela cells. The conjugate of IVIG and MTX showed potent and selective cytotoxicity to macrophages in vitro. [source] The extraordinary ligand binding properties of human serum albuminIUBMB LIFE, Issue 12 2005Mauro Fasano Abstract Human serum albumin (HSA), the most prominent protein in plasma, binds different classes of ligands at multiple sites. HSA provides a depot for many compounds, affects pharmacokinetics of many drugs, holds some ligands in a strained orientation providing their metabolic modification, renders potential toxins harmless transporting them to disposal sites, accounts for most of the antioxidant capacity of human serum, and acts as a NO-carrier. The globular domain structural organization of monomeric HSA is at the root of its allosteric properties which are reminiscent of those of multimeric proteins. Here, structural, functional, biotechnological, and biomedical aspects of ligand binding to HSA are summarized. [source] In vivo production of recombinant protein by a baculovirus vector inoculated perorally to the prefinal instar larvae of Bombyx mori L. (Lep., Bombycidae) aided by an optical brightener, Tinopal UNPA-GXJOURNAL OF APPLIED ENTOMOLOGY, Issue 9 2002T. Arakawa Host larvae were fed a diet containing 0.3% (w/w) Tinopal on day 1 in the 4th instar and then fed a diet contaminated by budded particles of NPV (1.0 × 106 TCID50 U/larva) that was pathogenic to B. mori (BmNPV) on day 2 (inoculation schedule 1). Another set of host larvae was fed a diet containing BmNPV budded particles (2.5 × 106 TCID50 U/larva) together with 0.3% (w/w) Tinopal on day 1 in the 4th instar (inoculation schedule 2). Host larvae treated by both schedules died of viral infection. The operation of schedule 2 is simpler than that of schedule 1, although the former required higher doses of the virus for satisfactory infection. We inoculated a baculovirus vector carrying human serum albumin (HSA) gene into 4th instar B. mori larvae by schedule 1. Recombinant HSA was detected in the homogenate of host larvae 4 days after inoculation. The peroral inoculation of BmNPV budded particles aided by Tinopal may thus lead to industrial pharmaceutical production using a baculovirus vector for large numbers of insect hosts. [source] A tandem MS precursor-ion scan approach to identify variable covalent modification of albumin Cys34: a new tool for studying vascular carbonylationJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 11 2008Giancarlo Aldini Abstract We developed a liquid chromatography electrospray ionisation multi-stage mass spectrometry (LC-ESI-MS/MS) approach based on precursor-ion scanning and evaluated it to characterize the covalent modifications of Cys34 human serum albumin (HSA) caused by oxidative stress and reactive carbonyl species (RCS) adduction. HSA was isolated and digested enzymatically to generate a suitable-length peptide (LQQCPF) containing the modified tag residue. The resulting LQQCPF peptides were identified by LC-ESI-MS/MS in precursor-ion scan mode and further characterized in product-ion scan mode. The product ions for precursor-ion scanning were selected by studying the MS/MS fragmentation of a series of LQQCPF derivatives containing Cys34 modified with different ,,,-unsaturated aldehydes and di and ketoaldehydes. We used a Boolean logic to enhance the specificity of the method: this reconstitutes a virtual current trace (vCT) showing the peaks in the three precursor-ion scans, marked by the same parent ion. The method was first evaluated to identify and characterize the Cys34 covalent adducts of HSA incubated with 4-hydroxy-hexenal, 4-hydroxy- trans -2-nonenal (HNE) and acrolein (ACR). Then we studied the Cys34 modification of human plasma incubated with mildly oxidized low-density lipoproteins (LDL), and the method easily identified the LQQCPF adducts with HNE and ACR. In other experiments, plasma was oxidized by 2,2'-azobis(2-amidinopropane) HCl (AAPH) or by Fe2+/H2O2. In both conditions, the sulfinic derivative of LQQCPF was identified and characterized, indicating that the method is suitable not only for studying RCS-modified albumin, but also to check the oxidative state of Cys34 as a marker of oxidative damage. Copyright © 2008 John Wiley & Sons, Ltd. [source] A fine match between the stereoselective ligands and membrane pore size for enhanced chiral separationAICHE JOURNAL, Issue 9 2009Honglei Wang Abstract A D,L -tryptophan separation factor of 12,15 and D -tryptophan yield of >95% have been successfully achieved through using human serum albumin (HSA) as the stereoselective ligand in an affinity ultrafiltration (UF) system. The obtained separation factor in this work is even higher than the intrinsic value of 8.5 of HSA. This synergism may arise from the fact that a fine match between the regular crystalline structure of HSA molecules and suitable pore size of membranes makes some HSA molecules be retained within the membrane cross-section, thus offering a second-stage binding opportunity for L -tryptophan molecules. Therefore, a simultaneous enhancement in separation factor and D -tryptophan yield has been fulfilled in this work. The feasibility of HSA regeneration after D,L -tryptophan separation has also been demonstrated through a series of pH adjustment experiments. This study reveals the applicability of HSA in affinity UF systems for chiral separation due to economization of material costs. © 2009 American Institute of Chemical Engineers AIChE J, 2009 [source] Mass spectrometric characterization of covalent modification of human serum albumin by 4-hydroxy- trans -2-nonenalJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 9 2006Giancarlo Aldini Abstract Several pieces of evidence indicate that albumin modified by HNE is a promising biomarker of systemic oxidative stress and that HNE-modified albumin may contribute to the immune reactions triggered by lipid peroxidation-derived antigens. In this study, we found by HPLC analysis that HNE is rapidly quenched by human serum albumin (HSA) because of the covalent adduction to the different accessible nucleophilic residues of the protein, as demonstrated by electrospray ionization mass spectrometry (ESI-MS) direct infusion experiments (one to nine HNE adducts, depending on the molar ratio used, from 1 : 0.25 to 1 : 5 HSA : HNE). An LC-ESI-MS/MS approach was then applied to enzymatically digested HNE-modified albumin, which permitted the identification of 11 different HNE adducts, 8 Michael adducts (MA) and 3 Schiff bases (SB), involving nine nucleophilic sites, namely: His67 (MA), His146 (MA), His242 (MA), His288 (MA), His510 (MA), Lys 195 (SB), Lys 199 (MA, SB), Lys525 (MA, SB) and Cys34 (MA). The most reactive HNE-adduction site was found to be Cys34 (MA) followed by Lys199, which primarily reacts through the formation of a Schiff base, and His146, giving the corresponding HNE Michael adduct. These albumin modifications are suitable tags of HNE-adducted albumin and could be useful biomarkers of oxidative and carbonylation damage in humans. Copyright © 2006 John Wiley & Sons, Ltd. [source] | ||||||||||||||||||||||||||||||||||