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Isolation of avidin and lysozyme from egg albumenDurance, Timothy Douglas January 1987 (has links)
A single column cation exchange method was developed which allowed simultaneous recovery of lysozyme and avidin from undiluted egg white using a unique elution sequence which involved accumulation of avidin on the column through several cycles of egg white application and lysozyme elution. Lysozyme was recovered with higher yields than reported for the isoelectric precipitation methods often used in the industry (86% vs 60 - 80%) and in high purity. Avidin recovery was also as good or better than that of previously reported ion exchange methods (74 - 80% vs 17 -80%). The purity of the avidin fraction (up to 40.9%) was superior to other reported primary avidin fractions. Avidin was shown to have a greater potential for both electrostatic and hydrophobic interactions with Duolite C-464 than lysozyme but under the conditions of this separation, electrostatic interactions were dominant.
Secondary purification of avidin by carboxymethyl celluose cation exchange (CMC), gel filtration, metal chelate interaction chromatography (MCIC), aliphatic hydrophobic interaction chromatography (HIC), and Phenyl-Sepharose interaction chromatography (PSIC) each resulted in considerable increase in avidin purity. In terms of resin capacity, yields, and avidin purity however, CMC ion exchange was superior. A comparison of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) andnative protein electrophoresis profiles gave clear evidence of protein - protein interaction between avidin and lysozyme in partially purified avidin preparations. This interaction may also occur between the native proteins in the egg white, but has not been demonstrated with certainty.
The molar ratio of avidin to available biotin binding sites was estimated by 5 methods. For highly purified avidin samples the hydroxy azo benzoic acid (HABA) method proposed by Green (1965) was superior. A new method, utilizing an immobilized biotin column, which did not require extensive purification of avidin was found to give similar results.
Finally, a highly sensitive assay of proteins bound to nitrocellulose membranes was developed which was capable of quantifying as little as 0.12 µg of protein. Membrane bound proteins were labeled with peroxidase via a biotin - avidin linkage as previously reported and bound peroxidase activity was related to initial protein content. The method was applicable to determination of the relative concentrations of different protein bands on Western blots of electrophoresis gels. / Land and Food Systems, Faculty of / Graduate
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Development of an avidin and C-reactive protein electrochemical immunosensorHennessey, Hooman. January 2006 (has links)
It has recently been shown that elevated C-reactive protein (CRP) levels are associated with a blunted systemic endothelial vasodilator function, indicative of a systemic inflammatory response. It has also been recognized that inflammation may contribute to all stages of the atherosclerotic process. Several prospective studies have shown that the level of CRP is a strong predictor of future myocardial dysfunction, stroke, peripheral arterial disease, and vascular death among individuals without known cardiovascular disease. CRP is also found in association with the senile plaques and neurofibrillary tangles of Alzheimer disease. Hence, determination of the blood serum levels of CRP is of a great clinical importance. / This thesis discusses the results on the development of two electrochemical immunosensors: (i) the avidin (model) immunosensor, and (ii) the CRP immunosensor. The suitability of using a range of electrochemical techniques in probing antibody-antigen interactions was also investigated. / It was shown that a gold working electrode surface could be successfully modified by covalent binding of NHS-biotin to a self-assembled-monolayer of cystamine dihydrochloride, resulting in the construction of the avidin immunosensor. Avidin could then bind to this immunosensor (biotinated electrode) and detected using the electrochemical techniques of cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), differential pulse voltammetry (DPV) and chronoamperometry (CA). It was also shown that polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) can be used to qualitatively and quantitatively characterize avidin bound to the immunosensor. The results demonstrated that all experimental techniques used were suitable in characterizing with high sensitivity the avidin-biotin interaction, and the amount of avidin in solution. The avidin calibration curves showed high linearity in concentrations ranging from 10-10 to 5 x 10-7 M. AFM imaging confirmed that avidin forms clusters upon binding to the immunosensor, with the cluster size ranging between 28 and 33 nm. The avidin-biotin binding constant was determined to be 3.09 x 1012 M-1 . It was also determined that the avidin-biotin equilibrium is reached in ca. 20 minutes. / The CRP immunosensor was then designed on the basis of the avidin immunosensor architecture. Using a range of electrochemical techniques and PM-IRRAS, it was demonstrated that a gold electrode surface could be functionalized by CRP antibodies, covalently attached to the surface through a duplex cystamine/glutaraldehyde layer. This architecture represents the CRP immunosensor. It was then shown that CRP antigen specifically binds to the CRP immunosensor (i.e. the immobilized CRP antibody). This interaction could be characterized with high sensitivity using the electrochemical techniques of CV, EIS, DPV and CA. The CRP concentration range investigated was 10-14 to 10-8 M. A linear calibration plot was obtained. The CRP antibody-antigen binding constant was determined to be 3+/-1 x 108 M-1. The corresponding binding equilibrium is reached in ca. 10 minutes. The results show that the developed CRP immunosensor is a good candidate for further research towards developing a commercial CRP immunosensor.
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The Effect of Avidin Injected Intraperitoneally on the Course of Leukemia in the Mouse / The Effect of Avidin Injected Peritoneally on the Course of Leukemia in the MouseJones, George Adams, III 01 1900 (has links)
The current work was undertaken to test the ability of avidin to affect the course of leukemia when administered intraperitoneally.
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Development of an avidin and C-reactive protein electrochemical immunosensorHennessey, Hooman. January 2006 (has links)
No description available.
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Model membranes grafted with long polymersNikolov, Vesselin Kirolov January 2004 (has links)
Wir untersuchen, welchen Einfluss die Verankerung von langen, hydrophilen Polymeren in Lipidmembranen auf deren elastische Eigenschaften ausübt. Theoretisch werden zwei Grenzbereiche für die spontane Krümmung der Membran erwartet: <br />
i) bei kleinen Oberflächenkonzentrationen des Polymers (Pilzgebiet) sollte die spontane Krümmung linear von der Oberflächendichte des verankerten Polymers abhängen;<br />
ii) bei hoher Bedeckung (Bürstengebiet) sollte die Abhängigkeit quadratisch sein. Wir versuchen, Vorhersagen für das Bürstengebiet zu prüfen, indem wir die morphologischen Veränderungen beobachten, die bei Riesen (Giant)- Vesikeln hervorgerufen werden. <br />
<br />
Als lange Polymere verwenden wir fluoreszenzmarkierte λ-Phage DNA Moleküle, die durch eine Biotin-Avidin-Biotin Verbindung an biotinhaltigen Lipidvesikeln befestigt sind. Wir kontrollieren die Oberflächenkonzentration der Anker durch Variation der Menge an biotinhaltigem Lipid in der Membran. Die Menge der an der Membran verankerten DNA wird durch Fluoreszenzmessungen quantifiziert. Änderungen in den elastischen Eigenschaften der Membran bei Anbindung der DNA, werden über eine Analyse der Vesikel-Fluktuationen kontrolliert. Die spontane Krümmung der Membran steigt mit der Oberflächenbeladung. Bei höheren Verankerungen bilden die Vesikel Knospen (budding). Die Größe der Knospen kann ebenfalls zur Bestimmung der Krümmung der Membran verwendet werden. Der Einfluss auf die Biegesteifigkeit ist Thema weiterer Untersuchungen. / We study the effect on the elastic properties of lipid membranes induced by anchoring of long hydrophilic polymers. Theoretically, two limiting regimes for the membrane spontaneous curvature are expected : <br />
i) at low surface polymer concentration (mushroom regime) the spontaneous curvature should scale linearly with the surface density of anchored polymers; <br />
ii) at high coverage (brush regime) the dependence should be quadratic. We attempt to test the predictions for the brush regime by monitoring the morphological changes induced on giant vesicles.<br />
<br />
As long polymers we use fluorescently labeled λ-phage DNA molecules which are attached to biotinylated lipid vesicles with a biotin-avidin-biotin linkage. By varying the amount of biotinylated lipid in the membrane we control the surface concentration of the anchors. The amount of anchored DNA to the membrane is quantified with fluorescence measurements. Changes in the elastic properties of the membrane as DNA grafts to it are monitored via analysis of the vesicle fluctuations. The spontaneous curvature of the membrane increases as a function of the surface coverage. At higher grafting concentrations the vesicles bud. The size of the buds can also be used to assess the membrane curvature. The effect on the bending stiffness is a subject of further investigation.
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Study on the production process of the recombinant his-tag streptavidinHuang, Chi-tien 14 February 2008 (has links)
In this study, we used E. coli strain BL21 (DE3) to express the recombinant protein his-tag streptavidin. To find out the optimal production conditions, we studied on the culture conditions, medium composition, induction conditions and the timing of induction. In the purification processes we tried to find out the difference between hydrophobic column and affinity column. We also tested the effect of heat treatment on the crude extract to increase the recombinant protein yield. The results showed that when cultured in LB medium, the optimal culture conditions of recombinant protein expression are 37¢XC, pH 6.0 to 7.0, and the induction temperature is 37¢XC. The best induction time is at late log phase or the early stationary phase when OD600 values reached to the ranged of 1.1 to 1.8. The inducer, IPTG concentration is 0.1 mM, which can also replaced with 2 mM lactose. The best production medium is TB medium. When cultured in 5 liters fermentor with optimal culture and induction condition, the highest recombinant protein yield could be 81.1 mg /L. To improve the purification process, we used a affinity chromatography. The purified high homogeneous recombinant protein had a high biotin binding activity up to 14 U / mg, and the recovery yield could be as high as 97% in comparing with the hydrophobic column was only 51%. When we treated the crude extract with 75 ¢J for 10 min, the biotin binding activity was 14.1 U / mg, but the recovery rate decreased to 64 %.
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Der Effekt von Sirolimus auf die reaktive Zellproliferation und Apoptose in einem humanen ex vivo Restenose-ModellZellmann, Svenja, January 2007 (has links)
Ulm, Univ., Diss., 2007.
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Non-covalent immobilisation of a ligand system : a new approach to affinity separationLiebenberg, Liesl Eileen 03 1900 (has links)
Thesis (MSc)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: Advances in pharmacology, biochemistry and biotechnology are increasingly dependant upon
affinity chromatography as a preferred separation technique for the purification and
characterisation of specific biomolecules.
In the past few years avidin-biotin technology has been widely and successfully used in the fields
of medicine, pharmacy, biology and biochemistry. The avidin-biotin complex (ABC) has been
used as a mediator for affinity chromatography, affinity cytochemistry, immunoassay,
histopathology, bioaffinity sensors, erosslinking and immobilisation studies.
The main reason for the popularity of the ABC and its growing usefulness in biotechnology is the
exceptionally high affinity (1015 M-l) and stability of the noncovalent interaction between avidin
and biotin. The use of the ABC is broadening as different biotin derivatives and
avidin-containing conjugates are becoming commercially available.
The aim of this work was to evaluate the usefulness of a plutonic" FI 08 and the ABC conjugate
to effect affinity separation. Towards this aim, the adsorption of plutonic" F108 onto
hydrophobic polysulphone membrane surfaces was studied. This information was used to
determine the theoretical maximum amount of pluronic" FI08 that will adsorb onto a unit
surface area of the membrane. It is known that the polypropylene oxide (PPO) centre block ofthe
pluronic" F I08 surfactant molecule governs the concentration of pluronic" F I 08 molecules that
will adsorb onto a given hydrophobic surface. If the maximum coating concentration of
plutonic" FI08 is known, one can assume that the maximum coating concentration of any
pluronic derivative, with the same PPO centre block size, will be the same. Adsorption studies
were carried out, the Langmuir adsorption isotherm was determined, and subsequently the
fractional coating was calculated.
The end-groups of plutonic" FI08 were modified as follows and the substituted pluronic was
adsorbed onto a membrane that was to act as the solid support matrix in the development of an
affinity system: Amino pluronic was synthesised by first tosylating pluronic" FI08, followed by azidation with NaN3 then reduction with LiAI~. The synthesised amino pluronic was then
biotinylated using N-hydroxysuccinimide biotin ester. The suitability of this synthetic route was
first assessed on a model compound, 2-methoxyethylamine, and validated by NMR (Nuclear
Magnetic Resonance) spectroscopy. The synthetic protocol was then used to derivatise the larger
pluronic molecule.
The affinity system was tested on two different hydrophobic surfaces: polystyrene and
polysulphone membranes (PSMs). Avidin-conjugated horseradish peroxidase was obtained and
used to interact with the immobilised biotin. The enzymatic reaction of the coupled peroxidase
converted the substrate, 2, 2'-azino-di-(3-ethyl-benzthiazoline-6-sulphonic acid) (ABTS) to a
coloured product. The colour developed is proportional to the amount of biotin that was
immobilised on the hydrophobic surfaces studied.
Non-covalent immobilisation of the synthesised biotin-pluronic molecule was successfully
obtained onto the hydrophobic polystyrene as well as the polysulphone membrane surfaces. / AFRIKAANSE OPSOMMING: Vooruitgang in die farmakologie, biochemie en biotegnologie word al meer afhanklik van
affiniteits chromatografie as die verkose tegniek vir die suiwering en karaterisering van
spesifieke biomolekules.
Oor die afgelope jare het die avidien-biotien tegnologie homself as baie bruikbaar bewys in die
mediese, farmakologiese, biologiese en biochemiese velde. Toepassings waar die avidien-biotien
kompleks betrokke was sluit in die toepassing as 'n mediator vir affiniteits chromatografie,
affiniteits sitologie, immuno bepalings, histopatologie, bioaffiniteits sensors sowel as
kruisbinding en immobiliserings studies en vele meer.
Die hoofrede vir die gewildheid van die avidien-biotien kompleks en die groeiende bruikbaarheid
in die biotegnologie is die buitengewone hoë affiniteit (l015 M-I
) en stabiliteit van die
nie-kovalente interaksie tussen avidien en biotien. Die toepassingsveld van die avidien-biotien
kompleks word wyer met die verskeidenheid biotien derivate en avidien-bevattende konjugate
wat kommersiëel beskikbaar is.
Die doel van die werk wat hier gedokumenteer word is om die bruikbaarheid van Plutonic" FI08
en die avidien-biotien kompleks, vir gebruik in 'n affiniteits chromatografie sisteem, te evalueer.
Om hierdie doel te bereik is die adsorpsie van Pluronic" FI08 aan hidrofobiese polisulfoon
membraan oppervlaktes bestudeer. Die eksperimentele data wat gegenireer is, is gebruik om die
teoretiese maksimum hoeveelheid Pluronic wat per eenheids oppervlakte membraan adsorbeer te
bepaal. Dit is reeds bekend dat die polipropileen (PPO) middel blok van die Pluronic emulgant
die konsentrasie van die geadsorbeerde Pluronic molekules op 'n gegewe hidrofobiese
oppervlakte bepaal. Indien die maksimum bedekkingskonsentrasie VIr maksimum
oppervlakbedekking van Plutonic" FI08 bekend is, kan teoreties aanvaar word dat die
bedekkingskonsentrasie vir enige Pluronic derivaat met dieselfde grootte PPO blok dieselfde sal
wees. Adsorpsiestudies was uitgevoer om die Langmuir adsorpsie isoterm te bepaal.
Daaropvolgend was die fraksionele bedekking bereken. Amino-pluronic was gesintetiseer deur die eindpunte van Pluronic te derivatiseer. Hierdie
Pluronic derivaat was gevolglik geadsorbeer aan 'n membraan wat gedien het as die soliede
oppervlakte vir die ontwikkeling van 'n affiniteits chromatografie sisteem.
Amino-pluronic was gesintetiseer deur Pluronic eers te tosileer en daarna te asideer met NaN3 en
laastens te reduseer met LiAI~. Die produk was gebiotinileer deur gebruik te maak van
N-hidroksisuksinimied-biotien-ester. Die bruikbaarheid van hierdie sintetiese roete is eers bepaal
deur van 'n model verbinding, 2-metoksiëtielamien, gebruik te maak en dit met behulp van KMR
(Kern Magnetiese Resonans) spektroskopie te karakteriseer.
Die affiniteits sisteem is getoets op twee verskillende hidrofobiese oppervlaktes naamlik
polistireen en polisulfoon membraan oppervlaktes. Avidien gekonjugeerd met 'n peroksiedase
ensiem is gebruik om met die geïmmobiliseerde biotien te assosieer. Die ensiematiese reaksie
van die gekoppelde peroksiedase het die substraat
2, 2' -azino-di-(3-etiel-benzthiazolien-6-sulfoonsuur) (ABTS) omgesit na 'n gekleurde produk,
waar dit teenwoordig is. 'n Reeks wasstappe is gebruik om die gemodifiseerde peroksidase
ensiem wat nie aan die hidrofobiese oppervlakte gekoppel nie, weg te spoel. Hierdeur is die mate
van binding aan die hirofobiese oppervlakte gekwantifiseer deur die kleur te kwantifiseer wat
ontwikkelomdat die kleurontwikkeling direk proporsioneel is aan die hoeveelheid peroksidase
wat nog aan die membraan gekoppel is.
Nie-kovalente immobilisasie van die gesintetiseerde biotien-pluronic molekule is suksesvolop
beide die hidrofobiese polistireen oppervlakte sowel as die polisulfoon membraan verkry.
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Biotinylation and high affinity avidin capture as a strategy for LC-MS based metabolomicsRhönnstad, Sofie January 2010 (has links)
<p>Metabolites, small endogenous molecules existing in every living cell, tissue or organism, play a vital role for maintaining life. The collective group of all metabolites, the metabolome, is a consequence of the biochemistry and biochemical pathways that a cell or tissue uses to promote survival. Analysis of the metabolome can be done to reveal changes of specific metabolites which can be a manifestation, a reason or a consequence of for example a disease. The physical chemical diversity amongst these components is tremendous and it poses a large analytical challenge to measure and quantify all of them. Targeting sub groups of the metabolome such as specific functional classes has shown potential for increasing metabolite coverage. Group selective labeling with biotin-tags followed by high affinity avidin capture is a well established purification strategy for protein purification.</p><p>The purpose with this project is to explore if it is possible to transfer the avidin biotin approach to metabolomics and use this method for small molecules purification. Specifically, this investigation aims to see if it is achievable to make a biotinylation of specific functional groups, to increase the sensitivity through reduction of sample complexity in liquid chromatography mass spectrometry metabolomics analyses after high affinity avidin capture. By purifying the analyte of interest and thereby reducing the sample complexity there will be a reduction in ion suppression. The aim is to increase the analytical sensitivity through a reduction in ion suppression during liquid chromatography mass spectrometry analysis.</p><p>Delimitations have been done to only investigate the possibility to obtain a biotinylation of primary amines and amides. As model compounds phenylalanine, spermidine, histamine and nicotinamide have been selected.</p><p>The result from this study indicates that it is possible to increase metabolite coverage through biotin labeling followed by high affinity avidin capture. It is a gain in analytical sensitivity of selected model compounds when comparing biotinylation strategy with a control nonbiotinylation approach in a complex sample. A broader study of additional model compounds and a method development of this strategy are necessary to optimize a potential future method.</p>
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Non-Covalent Selection Methodologies Utilizing Phage DisplayMeyer, Scott C. January 2007 (has links)
In nature, non-covalent interactions are as important and dynamic as they are elusive. As such, the study of non-covalent interactions both in vivo and in vitro has proven to be challenging. Given the potential benefits of elucidating protein-protein, ligand-receptor, and other biologically relevant interactions, the development of methodologies for the study of non-covalent interactions is an attractive goal.Biologically encoded protein and peptide libraries that connect the genotypic information with the expressed phenotype have emerged in recent years as powerful methods for studying non-covalent interactions. One of the quintessential platforms for the creation of such libraries is phage display. In phage display, the connection between genetic information and the corresponding protein allows for the iterative isolation and amplification of library members that possess a desired function. Hence, an in vitro selection can be used to isolate epitopes that bind to desired targets or display specific attributes.We have sought to develop novel phage display methodologies that have the potential to expand the scope of this in vitro selection platform. Specifically, we developed a method for the non-covalent attachment of a small molecule ligand to a cyclic peptide library. This system localizes the phage display library to the ligand binding site, thus allowing for the translation of the selected cyclic peptides to a covalently tethered bivalent inhibitor.The first class of biological molecules that we chose to target with our methodology is the biologically and therapeutically important class of enzymes called protein kinases. In the first demonstration of this strategy, we were able to isolate cyclic peptide ligands for the model kinase PKA (cAMP-dependent protein kinase), which were subsequently translated to a bivalent inhibitor. This inhibitor showed both increased affinity and selectivity for PKA in relation to other protein kinases.In a separate project, we sought to develop a method for the isolation of small molecule-responsive mutants of a well-characterized protein scaffold from a phage display library. During these investigations, we discovered interesting homologous single-point mutations of the protein that resulted in large spherical oligomers that may mimic species relevant to the study of protein misfolding diseases such as Alzheimer's.
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