Development of a synthetic affinity membrane for the purification of recombinant maltose binding proteins
Date
2008-12
Authors
Asongwe, Lionel Ateh Tantoh
Journal Title
Journal ISSN
Volume Title
Publisher
Stellenbosch : Stellenbosch University
Abstract
The aim of this project was to fabricate a new affinity membrane-based system that is biospecific
and biocompatible, and which could be used as an adsorption matrix for the
immobilization of the recombinant protein maltose binding protein human estrogen receptor
alpha ligand binding domain (MBP-hER LBD). The viability of the affinity membrane system
(AMS) for the detection of estrogenic compounds (ECs) in drinking water, using affinity
principles was determined. This affinity separation was based on the interaction between the
analyte 17 -estradiol (E2) and the recombinant protein MBP-hER LBD. The MBP-hER LBD
was immobilized on a solid matrix support. The alpha human estrogen hormone receptor
(hER ) was used to test for the binding affinity of the fusion protein to a ligand, radiolabelled E2.
Each component of this bioaffinity system, from the membrane matrix to the
expression/purification of the bioligand, and raising of antibodies against the purified bioligand,
was studied with the aim of producing a well-characterized system with the following
advantages: robust in nature, cost effective and high loading capacity.
Specifically, this study describes:
1. Expression of the bioligand maltose-binding protein (MBP) to be used as an affinity
ligand for immobilization onto a solid membrane matrix.
2. Expression of MBP as a fusion protein to the human estrogen receptor alpha ligand
binding domain (hER LBD).
3. The affinity purification of biospecific bioligands (MBP and MBP-hER LBD) using a
one-step affinity purification system with amylose forming the solid phase of the affinity
chromatographic column.
4. Generation of anti MBP-hER LBD antibodies to be used for the characterization of the
bioligands by means of western blotting.
5. The fabrication and characterization of a flat-sheet membrane as a model affinity-matrix.
6. Developing an affinity immobilization protocol for the immobilization of the bioligand
onto the affinity membrane (AM) matrix.
7. Quantitative analysis of the immobilized bioligand present on the surface of the
membrane matrix using tritiated E2. The recombinant protein (MBP-hER LBD) was successfully expressed and purified to form a
bio-specific ligand for its immobilization onto a cellulose acetate (CA)/amylose functionalized
affinity membrane. Polyclonal antibodies were successfully raised against the purified
recombinant protein. The anti-MBP-hER LBD antibodies were subsequently used as a potential
‘marker’ to confirm the immobilization of the recombinant protein onto the CA/amylose
functionalized membrane. Attempts to utilize the protein-coated membrane for the selective
recovery of E2 were, however, unsuccessful.
Description
Thesis (MSc (Biochemistry))--Stellenbosch University, 2008.
Keywords
Maltose binding protein, Endocrine disrupting compounds, Affinity chromatography, Recombinant proteins, Cellulose acetate/amylose affinity membrane (CA/amylose affinity membrane), Dissertations -- Biochemistry, Theses -- Biochemistry