O'Brien, Susan Mary; (1996) The separation of recombinant proteins by metal-chelating magnetic affinity supports. Doctoral thesis (Ph.D), UCL (University College London).

Text The_separation_of_recombinant_.pdf Download (14MB)

Abstract

The introduction of selective protein adsorption early in the downstream process has the potential to reduce the overall number of steps and increase efficiency and yield. However, the conventional format of a packed column of porous adsorbent is unsuitable for processing particulate containing feed streams as the matrix becomes clogged with trapped solids. There is evidence that non-porous supports are less prone to fouling and are easier to clean (Munro et al. 1977; Hailing and Dunnill, 1979) and are therefore potentially more useful in protein purification from fouling feedstreams. This work describes the preparation, characterisation and use of non-porous magnetic metal chelator adsorbents for the selective recovery of native (haem protein family) and recombinant metal-binding proteins from crude liquors. Non-porous, micron-sized (0.5-1.5 μm), magnetic iron oxide particles were functionalised with metal chelating iminodiacetic acid (IDA) ligands to produce a high capacity pseudoaffinity support. In magnetic affinity adsorption the covalently bound chelating ligand binds to its target protein in solution and the resulting complex is removed from suspension by the application of a magnetic field. The interaction is carried out in free solution and introduction at the start of the purification process renders pre-treatment steps such as clarification and concentration unnecessary and in doing so reduces costs and increases yield. The coating and derivatisation methods resulted in supports with a high level of substitution and low non-specific binding while retaining a high effective surface area for target protein capture (~ 100 m2/g). Supports were optimised with respect to ligand density (60 μmoles Cu2+/g), specific protein binding capacity (200 mg/g), and absence of nonspecific binding. Selectivity and interaction strength of magnetic chelator particles were assessed using a set of native proteins with known behaviour towards immobilised metal chelates. A recombinant bacteriophage T4 lysozyme carrying a polyhistidine tail at its C-terminus was purified in a single step to 94% purity from crude E. coli cell extract using IDA-Cu2+ magnetic chelator supports. By comparison, single step purification by Cu2+-charged IDA agarose supports achieved only 81% purity. Magnetic separation resulted in an overall yield of 82% which corresponded to a purification factor of 4.5.

Download activity - last month

Export as CSVXMLJSON

Download activity - last 12 months

Export as JSONXMLCSV

Downloads by country - last 12 months

Export as JSONXMLCSV

Archive Staff Only

View Item