Development of a synthetic affinity membrane for the purification of recombinant maltose binding proteins
Thesis (MSc (Biochemistry))--Stellenbosch University, 2008.
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.