Bioaffinity separation using ligand-modified pluronic and synthetic membranes

Govender, Selvakumaran (Stellenbosch : University of Stellenbosch, 2011-10)

Thesis (PhD)--University of Stellenbosch, 2005.

Thesis

ENGLISH ABSTRACT: A new membrane based affinity separation system that is bio-specific, biocompatible, well characterised and capable of being regenerated or re-used is described. The amphiphilic non-ionic surfactant Pluronic® F108, was covalently derivatised to form two novel bioligands (Pluronic-Biotin and Pluronic-DMDDO) for the bio-specific immobilisation of avidin conjugated proteins and histidine tagged proteins respectively. Pluronic was also used to non-covalently functionalise nonporous membranes for ligand attachment and to simultaneously shield the surfaces from non-specific protein adsorption. Each component of this bioaffinity system (from the membrane matrix to the elution/desorption of the ligate/ligand system) was studied with the aim of producing a well characterised system and key quantitative data for the development of a robust, reliable, re-usable and scalable technology. Specifically, this study describes: 1. The fabrication and partial characterisation of nonporous planar and capillary membranes as model affinity matrices. 2. The development and evaluation of a robust protocol for solvent desorption and accurate colorimetric quantification of Pluronic® F108 and its derivatives. 3. Interfacial analysis of Pluronic adsorption onto nonporous affinity membranes, including the direct solid-state analysis of model, halogenated Pluronic derivatives using nuclear microprobe analysis. 4. Development of a surfactant based protocol for affinity membrane regeneration and re-use. 5. Specific bioaffinity immobilisation of avidin conjugated peroxidase onto biotinylated membranes in the presence of model protein foulants. 6. Cloning and expression of C-terminal hex-histidine tagged human cytochrome b5 into the bacterial expression system E. coli BL-21 DE3. 7. Development and characterisation of an immobilised metal affinity membrane system for metal chelation (Ni2+, Cu2+ and Zn2+) using a new chelator Pluronic- N,N-dicarboxymethyl-3,6-diazaoctanedioate and the bio-specific immobilisation of N-terminal hex-histidine tagged pantothenate kinase.

AFRIKAANSE OPSOMMING: 'n Nuwe membraan-gebaseerde affiniteitskeidingsisteem word beskryf wat biospesifiek, bioversoenbaar en goed gekarakteriseer is, en geregenereer of hergebruik kan word. Die amfifiliese nie-ioniese surfaktant Pluronic is kovalent gederivatiseer om twee nuwe bioligande (Pluronic-Biotien en Pluronic-DMDDO) te vorm vir biospesifieke immobilisering van proteïnligate. Pluronic is ook gebruik om nie-poreuse membrane niekovalent te funksionaliseer vir ligandaanhegting en om hulle oppervlaktes teen niespesifieke proteïen-adsorbsie af te skerm. Elke komponent van hierdie bioaffiniteitsisteem (van die membraanmatriks tot die uitwas/desorpsie van die ligaat/ligand sisteem) is ondersoek met die doel om 'n goed-gekarakteriseerde sisteem te produseer en om kwantitatiewe data te genereer vir die ontwikkeling van 'n robuuste, betroubare, herbruikbare en opskaleerbare tegnologie. Hierdie studie beskryf spesifiek: 1. Die fabrisering en gedeeltelike karakterisering van nie-poreuse planêre en kapillêre membrane as model affiniteitsmatrikse. 2. Die ontwikkeling en evaluering van 'n robuuste protokol vir oplosmiddel desorpsie en akkurate kolorimetriese kwantifikasie van Pluronic® F108 en afgeleides daarvan. 3. Intervlakanalises van Pluronic adsorpsie op nie-poreuse affiniteitsmembrane, insluitend die direkte vastetoestand analise van model ligand-gemodifiseerde Pluronic deur die gebruik van kern-mikrosonde analise. 4. Ontwikkeling van 'n surfaktant-gebaseerde protokol vir affiniteitsmembraan regenerering en hergebruik. 5. Spesifieke bioaffiniteitsimmobilisering van avidien-gekonjugeerde peroksidase op gebiotinileerde membrane in die teenwoordigheid van model bevuilende proteïne. 6. Klonering en uitdrukking van C-terminaal hex-histidien geëtiketeerde menslike sitochroom b5 in die bakteriële uitdrukkingsisteem E. coli BL-21 DE3. 7. Ontwikkeling en karakterisering van 'n geïmmobiliseerde metaalaffiniteitsmembraansisteem vir metaalchelering (Ni2+, Cu2+ en Zn2+) met behulp van die nuwe cheleerder Pluronic-N,N-dikarboksimetiel-3,6- diasaoktaandioaat en die bio-spesifieke immobilisering van N-terminaal hexhistidiengeëtiketerde pantotenaatkinase.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/16516
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