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

Lipin, D. I., Lua, L. H. L. and Middelberg, A. P. J. (2008) 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. Journal of Chromatography A, 1190 1-2: 204-214. doi:10.1016/j.chroma.2008.03.032


Author Lipin, D. I.
Lua, L. H. L.
Middelberg, A. P. J.
Title 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
Journal name Journal of Chromatography A   Check publisher's open access policy
ISSN 0021-9673
Publication date 2008-05-09
Year available 2008
Sub-type Article (original research)
DOI 10.1016/j.chroma.2008.03.032
Open Access Status
Volume 1190
Issue 1-2
Start page 204
End page 214
Total pages 11
Editor Poole, C. F.
Dorsey, J. G.
Giese, R. W.
Place of publication Netherlands
Publisher Elsevier BV
Language eng
Subject C1
100799 Nanotechnology not elsewhere classified
100302 Bioprocessing, Bioproduction and Bioproducts
860899 Human Pharmaceutical Products not elsewhere classified
03 Chemical Sciences
0301 Analytical Chemistry
Abstract Polyomavirus VP1 protein in pentamer form was expressed in E. call 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: 5-10 nm; 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.
Keyword Aggregate
Field-Fow-Fractionation
Glutathione-S-Transferase
Light Scattering
Particle
Protein
Soluble Aggregate
Virus
Q-Index Code C1
Q-Index Status Confirmed Code
Institutional Status UQ

Document type: Journal Article
Sub-type: Article (original research)
Collections: Excellence in Research Australia (ERA) - Collection
Australian Institute for Bioengineering and Nanotechnology Publications
 
Versions
Version Filter Type
Citation counts: TR Web of Science Citation Count  Cited 27 times in Thomson Reuters Web of Science Article | Citations
Scopus Citation Count Cited 29 times in Scopus Article | Citations
Google Scholar Search Google Scholar
Created: Fri, 17 Apr 2009, 18:34:11 EST by Amanda Lee on behalf of Aust Institute for Bioengineering & Nanotechnology