Affinity purification of viral protein having heterogeneous quaternary structure: Modeling the impact of soluble aggregates on chromatographic performance

Lipin, D.I., Raj, A., Lua, L.H.L. and Middelberg, A.P.J. (2009) Affinity purification of viral protein having heterogeneous quaternary structure: Modeling the impact of soluble aggregates on chromatographic performance. Journal of Chromatography A, 1216 30: 5696-5708. doi:10.1016/j.chroma.2009.05.082


Author Lipin, D.I.
Raj, A.
Lua, L.H.L.
Middelberg, A.P.J.
Title Affinity purification of viral protein having heterogeneous quaternary structure: Modeling the impact of soluble aggregates on chromatographic performance
Journal name Journal of Chromatography A   Check publisher's open access policy
ISSN 0021-9673
Publication date 2009-07-24
Year available 2009
Sub-type Article (original research)
DOI 10.1016/j.chroma.2009.05.082
Volume 1216
Issue 30
Start page 5696
End page 5708
Total pages 13
Editor J. G. Dorsey
Place of publication Netherlands
Publisher Elsevier BV
Collection year 2010
Language eng
Subject C1
Formatted abstract
Prokaryote-expressed polyomavirus structural protein VP1 with an N-terminal glutathione-S-transferase tag (GST-VP1) self-assembles into pentamer structures that further organize into soluble aggregates of variable size (3.4 × 102–1.8 × 104 kDa) [D.I. Lipin, L.H.L. Lua, A.P.J. Middelberg, J. Chromatogr. A 1190 (2008) 204]. The adsorption mechanism for the full range of GST-VP1 soluble aggregates was described assuming a dual-component model [T.Y. Gu, G.J. Tsai, G.T. Tsao, AICHE J. 37 (1991) 1333], with components differentiated by size, and hence pore accessibility, rather than by protein identity. GST-VP1 protein was separated into two component groups: aggregates small enough to access resin pores (LMW: 3.4 × 102–1.4 × 103 kDa) and aggregates excluded from the resin pores (HMW: 9.0 × 102–1.8 × 104 kDa). LMW aggregates bound to resin at a higher saturation concentration (29.7 g L−1) than HMW aggregates (13.3 g L−1), while the rate of adsorption of HMW aggregates was an order of magnitude higher than for LMW aggregates. The model was used to predict both batch and packed bed adsorption of GST-VP1 protein in solutions with known concentrations of HMW and LMW aggregates to Glutathione Sepharose HP resin. Asymmetrical flow field flow fractionation with UV absorbance was utilized in conjunction with adsorption experimentation to show that binding of HMW aggregates to the resin was strong enough to withstand model-predicted displacement by LMW aggregates. High pore concentrations of LMW aggregates were also found to significantly inhibit the diffusion rate of further protein in the resin pores. Additional downstream processing experimentation showed that enzymatic cleavage of LMW aggregates to remove GST tags yields more un-aggregated VP1 pentamers than enzymatic cleavage of HMW aggregates. This model can be used to enhance the chromatographic capture of GST-VP1, and suggests an approach for modeling chromatographic purification of proteins that have a range of quaternary structures, including soluble aggregates. ©2009 Elsevier B.V. All rights reserved.
Keyword Virus-like particle
Polyomavirus
VP1
Soluble aggregate
Affinity chromatography
Modeling
Glutathione-S-transferase
Asymmetrical flow field flow fractionation
VIRUS-LIKE PARTICLES
FIELD-FLOW FRACTIONATION
BIOSPECIFIC ADSORPTION
ESCHERICHIA-COLI
SIZE
BEDS
SIMULATION
DISPERSION
SEPARATION
LYSOZYME
Q-Index Code C1
Q-Index Status Confirmed Code

Document type: Journal Article
Sub-type: Article (original research)
Collections: 2010 Higher Education Research Data Collection
Australian Institute for Bioengineering and Nanotechnology Publications
 
Versions
Version Filter Type
Citation counts: TR Web of Science Citation Count  Cited 15 times in Thomson Reuters Web of Science Article | Citations
Scopus Citation Count Cited 15 times in Scopus Article | Citations
Google Scholar Search Google Scholar
Created: Fri, 04 Sep 2009, 10:26:48 EST