Thesis (PhD)--University of Stellenbosch, 2005. / 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.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/16516 |
| Date | 10 1900 |
| Creators | Govender, Selvakumaran |
| Contributors | Swart, P., Jacobs, E.P., Bredenkamp, M.W., University of Stellenbosch. Faculty of Science. Dept. of Biochemistry. |
| Publisher | Stellenbosch : University of Stellenbosch |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | xiv, various foliations : ill. |
| Rights | University of Stellenbosch |
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