Milburn, Philip Thomas; (1993) Optimization of Large Scale Virus-like Particle Purification. Doctoral thesis (Ph.D), UCL (University College London).

Text 10045565.pdf Download (11MB)

Abstract

Recombinant Ty virus-like particles (Ty-VLPs), expressed in the yeast Saccharomyces cerevisiae, have a number of potential applications, particularly as potent vaccines. In order to manufacture Ty-VLPs commercially it is essential that they can be efficiently purified on a large scale from the cells in which they are expressed. This thesis describes three studies which were carried out to provide a biochemical engineering foundation for the large scale purification of 5620 Ty-VLPs. A reversed phase (RP-) HPLC assay was developed to replace gel-scanning and immunoblotting for the measurement of 5620 Ty-VLPs. This assay was calibrated for quantitative measurement of 5620 Ty-VLPs, and its ability to measure 5620 Ty-VLPs quantitatively was verified. Compared to the previous assay methods, the RP-HPLC assay was simpler to carry out, faster, and of equal of better quantitative capability. It was however inferior in terms of sensitivity. Because 5620 Ty-VLPs are expressed as an intracellular product, cell disruption is required as a first step in 5620 Ty-VLP purification. The use of a high pressure homogenizer and a bead mill for this purpose was investigated. 5620 Ty-VLP release was most efficiently achieved with the homogenizer, and optimum conditions for release were determined. Freezing and thawing of the cells were found to increase their resistance to subsequent breakage in the homogenizer. The use of flocculating reagents for the clarification of yeast homogenate containing 5620 Ty-VLPs was investigated. Flocculation with sodium tetraborate (borax) selectively removed cell wall debris from the homogenate, leaving 5620 Ty-VLPs in the clarified liquor in high yield. Flocculation with polyethyleneimine (PEI) proved very effective for the selective removal of a range of non-protein contaminants from Baker's yeast homogenate. Soluble proteins remained in solution following flocculation, although high molecular weight proteinaceous material was removed. In agreement with this observation, 5620 Ty-VLPs were lost from solution when homogenate containing them was flocculated with PEI.

Download activity - last month

Export as JSONXMLCSV

Download activity - last 12 months

Export as CSVXMLJSON

Downloads by country - last 12 months

Export as JSONXMLCSV

Archive Staff Only

View Item