Wednesday, November 7, 2007 - 9:10 AM
386c

Quaternary Self-Association of Gst-Tagged Viral Proteins

D. I. Lipin, Centre for Biomolecular Engineering, Department of Chemical Engineering, The University of Queensland, Brisbane, Australia, Yap Pang Chuan, Centre for Biomolecular Engineering, Australian Institute of Bioengineering and Nanotechnology, The University of Queensland, Brisbane, Australia, L. Lua, Centre for Biomolecular Engineering, Australian Institue of Bioengineering and Nanotechnology, The University of Queensland, Brisbane, Australia, and A. P. J. Middelberg, Centre for Biomolecular Engineering, Australian Institue of Bioengineering and Nanotechnology, The University of Queensland, Brisbane, Australia.

Virus Like Particles (VLPs) are self-assembling nanoparticles with current and potential uses in the areas of vaccine production, gene therapy and drug delivery. Existing manufacturing processes largely seek to purify VLPs that have been pre-formed as complete nanoparticles in a cell. This limits control of VLP assembly and introduces a complex downstream nanoparticle separation challenge, as well as the need to devise innovative approaches to remove inadvertently encapsulated cellular contaminants. This study utilizes the paradigm of production and purification of less complex viral Lego blocks that are assembled into full VLPs in a cell-free reactor. The process begins with recombinant expression of the VLP major structural protein (polyomavirus VP1) with an N-terminal glutathione-s-transferase (GST) tag and purification from lysate in pentamer intermediate form by GST affinity chromatography. Enzymatic cleavage then removes the tag and individual pentamers are isolated by size exclusion chromatography (SEC). This study focused on the GST tagged intermediate protein (GST-VP1). Dynamic light scattering (DLS) and asymmetrical flow field flow fractionation with multiple angle light scattering (AFFFF-MALS) analysis of purified protein solutions indicated a previously unknown quaternary structure distribution. Analysis suggested the existence of macro-structures a few to several hundred pentamers in size. Formation of these natively multimeric protein structures is thought to be due to the occurrence of GST homo-dimerization driving pentamer association. Experimentation has shown that structures of all sizes are completely broken down by enzymatic cleavage into VP1 and GST, implying that the aggregation state retains the tertiary structure of the individual proteins. Stability studies involving these macrostructures have also shown constant changes in size distribution, potentially due to the reversible nature of the GST homo-dimerization reaction.

The multimeric nature of this protein intermediate presents several downstream processing benefits and challenges. The methods and understanding shown in this study will allow for a more thorough approach to deal with these challenges as well as to allow for better process validation to take place.