Quaternary size distribution of soluble aggregates of glutathione-S-transferase-purified viral protein as determined by asymmetrical flow field flow fractionation and dynamic light scattering.
Polyomavirus VP1 protein in pentamer form was expressed in E. coli and purified using glutathione-S-transferase (GST) affinity chromatography. Purified GST-tagged protein was found to exist as soluble aggregates with a size distribution of 1-52 tagged pentamers (340-1800 x 10(3)kDa), as determined by asymmetrical flow field flow fractionation with multiple angle light scattering (AFFFF-MALS). Aggregation did not inhibit tag removal by enzymatic cleavage, implying that the quaternary structure of the VP1 pentamers had been maintained. Elution gel filtration (EGF) was utilized to prepare a solution enriched with protein small enough to access resin pores (LMWe) as well as solution enriched with protein excluded from resin pores (HMWe). Material size distributions within both solutions were determined using AFFFF-MALS (radius of gyration LMWe: 5-10nm; HMWe: 10-35 nm) and dynamic light scattering (DLS) (hydrodynamic diameter LMWe: 10-90 nm; HMWe: 20-300 nm). DLS and AFFFF-MALS analysis of each fraction of affinity chromatography purified material identified the elution profiles of large and small aggregate structures. DLS readings of all fractions were significantly affected by the presence of high molecular weight aggregates, with Z-average hydrodynamic diameter values reflecting the mass ratio of large and small aggregate structures in a solution. The methods utilized in this study have the potential to be used during chromatographic purification of all proteins that exist as soluble aggregates to determine size distribution. The finding that GST-tagged viral proteins exist as soluble aggregates has implications for existing immunological studies that utilize them.
Lipin
Daniel I
DI
Centre for Biomolecular Engineering, Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, Queensland 4072, Australia.
Lua
Linda H L
LH
Middelberg
Anton P J
AP
D002850:Chromatography, Gel; D031521:Fractionation, Field Flow; D005982:Glutathione Transferase / Q000737:chemistry / Q000378:metabolism*; D008027:Light; D010316:Particle Size; D012542:Scattering, Radiation; D014764:Viral Proteins / Q000302:isolation & purification*
D014764:Viral Proteins; D005982:Glutathione Transferase